Mammalian cytokines; related reagents

ABSTRACT

Purified genes encoding a cytokine or composite cytokine from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding these molecules are provided. Methods of using said reagents and diagnostic kits are also provided.

[0001] This filing is a continuation-in-part of U.S. Utility patentapplication of U.S. Ser. No. 09/791,497, filed Feb. 22, 2001, which is acontinuation-in-part of U.S. Ser. No. 09/568,699, filed Sep. 8, 2000,and claims benefit from U.S. Provisional Patent Applications U.S. Ser.No. 60/146,581, filed Jul. 30, 1999; and U.S. Ser. No. 60/147,763, filedAug. 6, 1999, each of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

[0002] The present invention pertains to compositions related toproteins which function in controlling biology and physiology ofmammalian cells, e.g., cells of a mammalian immune system. Inparticular, it provides purified genes, proteins, antibodies, andrelated reagents useful, e.g., to regulate activation, development,differentiation, and function of various cell types, includinghematopoietic cells.

BACKGROUND OF THE INVENTION

[0003] Recombinant DNA technology refers generally to the technique ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

[0004] For some time, it has been known that the mammalian immuneresponse is based on a series of complex cellular interactions, calledthe “immune network”. Recent research has provided new insights into theinner workings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which will lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system disorders. Some of thesefactors are hematopoietic growth and/or differentiation factors, e.g.,stem cell factor (SCF) or IL-12. See, e.g., Mire-Sluis and Thorpe (1998)Cytokines Academic Press, San Diego; Thomson (ed. 1998) The CytokineHandbook (3d ed.) Academic Press, San Diego; Metcalf and Nicola (1995)The Hematopoietic Colony Stimulating Factors Cambridge University Press;and Aggarwal and Gutterman (1991) Human Cytokines Blackwell.

[0005] Lymphokines apparently mediate cellular activities in a varietyof ways. They have been shown to support the proliferation, growth, anddifferentiation of pluripotential hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages making up acomplex immune system. Proper and balanced interactions between thecellular components are necessary for a healthy immune response. Thedifferent cellular lineages often respond in a different manner whenlymphokines are administered in conjunction with other agents.

[0006] Cell lineages especially important to the immune response includetwo classes of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

[0007] Another important cell lineage is the mast cell (which has notbeen positively identified in all mammalian species), which is agranule-containing connective tissue cell located proximal tocapillaries throughout the body. These cells are found in especiallyhigh concentrations in the lungs, skin, and gastrointestinal andgenitourinary tracts. Mast cells play a central role in allergy-relateddisorders, particularly anaphylaxis as follows: when selected antigenscrosslink one class of immunoglobulins bound to receptors on the mastcell surface, the mast cell degranulates and releases mediators, e.g.,histamine, serotonin, heparin, and prostaglandins, which cause allergicreactions, e.g., anaphylaxis.

[0008] IL-12 plays a critical role in cell-mediated immunity (Gately etal. (1998); Trinchieri (1998); and Trinchieri (1995)). Its activitiesare triggered through a high-affinity receptor complex that gathers twoclosely related subunits, IL-12Rβ1 and β2 (Chua, et al. (1995); andPreskey et al. (1996b)). The p35 subunit has been suggested to bind to asecond a second soluble cytokine receptor called EBI3 (Devergne et al.(1997)). As yet no biological activity has been reported for thep35-EBI3 pair, however, pairings of IL-12 subunits or IL-12-likesubunits with other cytokines may provide information aboutcell-mediated immunity, e.g. T-cell regulation. Furthermore, thediscovery of receptors or receptor subunits for these heteromericcytokines will also provide information regarding immune regulation.

[0009] Research to better understand and treat various immune disordershas been hampered by the general inability to maintain cells of theimmune system in vitro. Immunologists have discovered that culturingthese cells can be accomplished through the use of T-cell and other cellsupernatants, which contain various growth factors, including many ofthe lymphokines.

[0010] From the foregoing, it is evident that the discovery anddevelopment of new lymphokines and their related receptors or receptorsubunits e.g., related to the IL-6/IL-12 cytokine family couldcontribute to new therapies for a wide range of degenerative or abnormalconditions, which directly or indirectly involve the immune systemand/or hematopoietic cells. In particular, the discovery and developmentof lymphokines which enhance or potentiate the beneficial activities ofknown lymphokines would be highly advantageous. The present inventionprovides new interleukin compositions, receptor subunits, and relatedcompounds, and methods for their use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the comparison between SEQ ID NO: 2 and the IL-D80variant polypeptide of SEQ ID NO: 6.

[0012]FIG. 2 shows a comparison of rodent IL-D80 (SEQ ID NO: 4) andvariant rodent IL-D80 (SEQ ID NO: 8) polypeptide sequences.

[0013]FIG. 3 shows a comparison of human IL-D80 (SEQ ID NO: 6) androdent, e.g., mouse IL-D80 (SEQ ID NO: 8)

SUMMARY OF THE INVENTION

[0014] The present invention is directed to mammalian, e.g., rodent,canine, feline, primate, interleukin numbered DNAX 80 (IL-D80; p28) andits biological activities. The present invention is also based upon thediscovery of the association of IL-D80 with the IL-12p40-like molecule,EBI3, and the binding of this composite cytokine to an IL-12Rβ2 subunithomologue known as WSX-1/TCCR. The IL-D80/EBI3 composite cytokine isalso known as IL-27. It includes nucleic acids coding for polypeptidesthemselves and methods for their production and use. The nucleic acidsof the invention are characterized, in part, by their homology tocomplementary DNA (cDNA) sequences disclosed herein, and/or byfunctional assays for growth factor- or cytokine-like activities, e.g.,IL-6/IL-12 family of cytokines (see Thomson (1998) The Cytokine Handbook3d ed., Academic Press, San Diego), applied to the polypeptides, whichare typically encoded by these nucleic acids. Methods for modulating orintervening in the control of a growth factor dependent physiology or animmune response are provided.

[0015] The present invention is based, in part, upon the discovery ofnew cytokine sequences exhibiting significant sequence and structuralsimilarity to the IL-6/IL12 family of cytokines. In particular, itprovides primate, e.g., human, and rodent, e.g., mouse, sequences.Functional equivalents exhibiting significant sequence homology will beavailable from other mammalian, e.g., cow, horse, and rat, mouse, andnon-mammalian species.

[0016] In various protein embodiments, the invention provides: asubstantially pure or recombinant IL-D80 polypeptide exhibiting identityover a length of at least about 12 amino acids to SEQ ID NO: 2, 4, 6, or8; a natural sequence IL-D80 of SEQ ID NO: 2, 4, 6, or 8; and a fusionprotein comprising IL-D80 sequence of SEQ ID NO: 2, 4, 6, or 8. Incertain embodiments, the segment of identity is at least about 14, 17,or 19 amino acids.

[0017] In other embodiments, the IL-D80 comprises a mature sequencecomprising the sequences from SEQ ID NO:2, 4, 6, or 8; or exhibits apost-translational modification pattern distinct from natural IL-D80; orthe polypeptide: is from a warm blooded animal selected from a mammal,including a primate; comprises at least one polypeptide segment of SEQID NO: 2, 4, 6, or 8; exhibits a plurality of amino acid residuefragments; is a natural allelic variant of IL-D80; has a length at leastabout 30 amino acids; exhibits at least two non-overlapping epitopeswhich are specific for a primate IL-D80; exhibits sequence identity overa length of at least about 20 amino acids to primate IL-D80; isglycosylated; has a molecular weight of at least 10 kD with naturalglycosylation; is a synthetic polypeptide; is attached to a solidsubstrate; is conjugated to another chemical moiety; is a 5-fold or lesssubstitution from natural sequence; or is a deletion or insertionvariant from a natural sequence. Preferred embodiments include acomposition comprising: a sterile IL-D80 polypeptide; or the IL-D80polypeptide and a carrier, wherein the carrier is: an aqueous compound,including water, saline, and/or buffer; and/or formulated for oral,rectal, nasal, topical, or parenteral administration. In fusion proteinembodiments, the protein can have: mature polypeptide sequence from SEQID NO:2, 4, 6, or 8; a detection or purification tag, including a FLAG,His6, or Ig sequence; and/or sequence of another cytokine or chemokine,including an IL-12.

[0018] Kit embodiments include those with an IL-D80 polypeptide, and: acompartment comprising the polypeptide; and/or instructions for use ordisposal of reagents in the kit.

[0019] In binding compound embodiments, the compound may have an antigenbinding site from an antibody, which specifically binds to a naturalIL-D80 polypeptide, wherein: the IL-D80 is a primate protein; thebinding compound is an Fv, Fab, or Fab2 fragment; the binding compoundis conjugated to another chemical moiety; or the antibody: is raisedagainst a peptide sequence of a mature polypeptide portion from SEQ IDNO:2, 4, 6, or 8; is raised against a mature IL-D80; is raised to apurified primate IL-D80; is immunoselected; is a polyclonal antibody;binds to a denatured IL-D80; exhibits a Kd of at least 30 _M; isattached to a solid substrate, including a bead or plastic membrane; isin a sterile composition; or is detectably labeled, including aradioactive or fluorescent label. Kits containing binding compoundsinclude those with: a compartment comprising the binding compound;and/or instructions for use or disposal of reagents in the kit. Oftenthe kit is capable of making a qualitative or quantitative analysis.Preferred compositions will comprise: a sterile binding compound; or thebinding compound and a carrier, wherein the carrier is: an aqueouscompound, including water, saline, and/or buffer; and/or formulated fororal, rectal, nasal, topical, or parenteral administration.

[0020] Nucleic acid embodiments include an isolated or recombinantnucleic acid encoding an IL-D80 polypeptide or fusion protein, wherein:the IL-D80 is from a primate; and/or the nucleic acid: encodes anantigenic peptide sequence of SEQ ID NO:2, 4, 6, or 8; encodes aplurality of antigenic peptide sequences of SEQ ID NO:2, 4, 6, or 8;exhibits identity to a natural cDNA encoding the segment; is anexpression vector; further comprises an origin of replication; is from anatural source; comprises a detectable label; comprises syntheticnucleotide sequence; is less than 6 kb, preferably less than 3 kb; isfrom a primate, including a human; comprises a natural full lengthcoding sequence; is a hybridization probe for a gene encoding theIL-D80; or is a PCR primer, PCR product, or mutagenesis primer. Theinvention also provides a cell, tissue, or organ comprising such arecombinant nucleic acid, and preferably the cell will be: a prokaryoticcell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell;a mammalian cell; a mouse cell; a primate cell; or a human cell.

[0021] Kit embodiments include those with such nucleic acids, and: acompartment comprising the nucleic acid; a compartment furthercomprising the IL-D80 protein or polypeptide; and/or instructions foruse or disposal of reagents in the kit. Typically, the kit is capable ofmaking a qualitative or quantitative analysis.

[0022] In certain embodiments, the nucleic acid: hybridizes under washconditions of 30° C. and less than 2M salt, or of 45° C. and/or 500 mMsalt, or 55° C. and/or 150 mM salt, to SEQ ID NO: 1, 3, 5, or 7; orexhibits identity over a stretch of at least about 30, 55, or 75nucleotides, to a primate IL-D80.

[0023] The invention embraces a method of modulating physiology ordevelopment of a cell or tissue culture cells comprising contacting thecell with an agonist or antagonist of a primate IL-D80. The method maybe where: the contacting is in combination with an agonist or antagonistof IL-12; or the contacting is with an antagonist, including a bindingcomposition comprising an antibody binding site which specifically bindsan IL-D80.

[0024] The invention further provides a composite cytokine (IL-27)comprising a plurality of segments of SEQ ID NO:2, 4, 6, or 8 and SEQ IDNO: 10. Also encompassed is an isolated or recombinant polynucleotideencoding the composite cytokine of said composite cytokine. Furtherprovided is a receptor subunit:ligand composition comprising a pluralityof polypeptide segments of SEQ ID NO:2, 4, 6, or 8, SEQ ID NO:10, andSEQ ID NO:12.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] All references cited herein are incorporated herein by referenceto the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

[0026] I. General

[0027] The present invention provides amino acid sequences and DNAsequences encoding various mammalian proteins, which are cytokines,e.g., which are secreted molecules which can mediate a signal betweenimmune or other cells. See, e.g., Paul (1997) Fundamental Immunology (3ded.) Raven Press, N.Y. The full length cytokines, and fragments, orantagonists will be useful in physiological modulation of cellsexpressing a receptor. It is likely that IL-D80 or IL-27 has eitherstimulatory or inhibitory effects on hematopoietic cells, including,e.g., lymphoid cells, such as T-cells, B-cells, natural killer (NK)cells, macrophages, dendritic cells, hematopoietic progenitors, etc. Inparticular, the IL-27 composite cytokine may play a role ininflammation, including, but not limited to ulcerative colitis,arthritis, etc. The proteins will also be useful as antigens, e.g.,immunogens, for raising antibodies to various epitopes on the protein,both linear and conformational epitopes.

[0028] A cDNA encoding IL-D80 was identified from various primate, e.g.,human, sequences of BACs of Chromosome 16. See, e.g., CIT987SK-A-575C2,and CIT987SK-A-761H5. The molecule was designated huIL-D80. A human ESThas been identified and described, human EST A1085007. A mouse ESTAA266872 has also been identified and described.

[0029] The primate, e.g., human, gene will encode a small solublecytokine-like protein, of about 216 amino acids (for SEQ ID NO: 2) orabout 243 amino acids (for SEQ ID NO: 6). See SEQ. ID. NOs: 1, 2, 5, and6. Exon boundaries are likely to correspond to about 219/220; 393/394;492/493; and 551/552 of SEQ ID NO:1. Coding segments corresponding tothose boundaries are particularly interesting. Translated amino acidsequence, which is encoded by nucleotides 193 to 918 of SEQ ID NO:1, isshown in SEQ ID NO: 2.

[0030] A predicted signal cleavage site may exist between about residues25-30 of SEQ ID NO: 2; helix A is predicted to run from about residues33-38 to about residues 54-59 of SEQ ID NO: 2; helix B is predicted torun from about residues 85-90 to about residues 111-116 of SEQ ID NO: 2;helix C is predicted to run from about residues 121-126 to aboutresidues 154-159 of SEQ ID NO: 2; and helix D is predicted to run fromabout residues 201-206 to about residues 228-233 of SEQ ID NO: 2.

[0031] SEQ ID NO: 5 shows a variant of IL-D80 and SEQ ID NO: 6 is theencoded polypeptide. FIG. 1 shows the comparison between SEQ ID NO: 2and the IL-D80 variant polypeptide of SEQ ID NO: 6. Structural motifsare as indicated above with the appropriate change in residue positions.

[0032] The corresponding rodent polynucleotide sequence of IL-D80 isshown in SEQ ID NO: 3. Exon boundaries are likely to run from about198/199; 360/361; 459/460; and 618/619. The predicted polypeptidesequence, which runs from about nucleotide 199 to 891 of SEQ ID NO: 3,is shown in SEQ ID NO: 4. The predicted signal cleavage site runs fromabout residue 16-21 of SEQ ID NO: 4; helix A is predicted to run fromabout residue 21-26 to about residue 41-46; helix B is predicted to runfrom about residue 72-77 to about residue 101-106; helix C is predictedto run from about residue 108-133 to about residue 141-146; and helix Dis predicted to run from about residue 185-190 to about residue 211-215.All postions refer to SEQ ID NO: 4. A variant rodent IL-D80polynucleotide sequence is shown in SEQ ID NO: 7 and the predictedpolypeptide sequence is shown in SEQ ID NO: 6. A comparison of rodentIL-D80 (SEQ ID NO: 4) and variant rodent IL-D80 (SEQ ID NO: 8)polypeptide sequences is shown in FIG. 2.

[0033] IL-D80 exhibits structural motifs characteristic of a member ofthe long chain cytokines belonging to the IL-6/1IL-12 family ofcytokines. The structural homology of IL-D80 to related cytokineproteins suggests related function of this molecule.

[0034] The IL-D80 cDNA sequences mature proteins with calculatedmolecular mass of 24.5 and 23.6 kDa. No N-glycosylation sites are foundin hIL-D80, but several O-glycosylation sites are predicted. MurineIL-D80 contains one potential N-glycosylation site (N85). Transientexpression of mp28 in the presence or absence of tunicamycin indicatedthat mp28 is indeed N-linked glycosylated. Both human and mouse IL-D80display an unusual sequence insertion in the predicted loop regionbetween helix C and D. In hIL-D80, the C-D loop contains a stretch of 13glutamic acid residues; mp28 displays 14 negatively charged residues inthis region, interrupted by one lysine residue. This highly chargedsequence has not been observed in any other helical cytokine and mostlikely will affect the biophysical properties of the protein insolution. Overall, human and mouse IL-D80 are 74% identical.

[0035] Comparison of the sequences will also provide an evolutionarytree. This can be generated, e.g., using the TreeView program incombination with the ClustaIX analysis software program. See Thompson,et al. Nuc. Acids Res. 25:4876-4882; and TreeView, Page, IBLS,University of Glasgow, e-mail rpage@bio.gla.ac.uk;http://taxonomy.zoology.gla.ac.uk.rod.treeview.html.

[0036] Co-transfection of human Epstein-Barr virus-induced gene 3 (EBI3;GenBank NM005755; Devergne, et al. (1996) J. Virol. 70:1143-1153; SEQ IDNOs: 9 and 10) cDNA and human IL-D80 cDNA leads to enhanced secretion ofIL-D80. IL-D80 co-immunoprecipitated with EBI3, and conversely, EBI3co-immunoprecipitated with IL-D80. This indicates that these twoproteins form a composite factor that either itself has biologicalfunctions (that neither protein has on its own) or EBI3 is used as ashuttle to release IL-D80 in the supernatant. Of note, EBI3 is alsoexpressed in vivo by activated antigen presenting cells (APCs) and atvery high levels by placental syncytiotrophoblasts. The presentinvention provides the first evidence that the IL-80D/EBI3 compositecytokine (IL-27) binds to an IL-12R-like subunit, WSX-1/TCCR (See, e.g.,GenBank AF265242; Chen, et al. (2000) Nature 407:916-920; SEQ ID NO: 11and 12).

[0037] Biologically, IL-27 is produced by antigen presenting cells(APCs). In contrast to other similar heterodimers made by APCs, i.e.,IL-12 (p35+p40) and IL-23 (p19+p40), kinetic analysis of IL-27 showedthat this composite cytokine is produced earlier in activation of APCs.Thus, IL-27 can be a potent adjuvant of a Th1 response.

[0038] The primary activity of IL-27 triggers rapid clonal expansion ofantigen specific naive human and mouse CD4+ T cells. Moreover, itpromotes Th1 polarization and IFNγ production of naïve CD4+ T cells.Mechanistically, these naive T cells are primed to response to IL-27 bythe production of this composite cytokine by the APCs which interactwith these cells. These activities of IL-27 are dependent onsimultaneous T cell receptor activation and occur in synergy with IL-12.

[0039] IL-D80 or IL-27 agonists, or antagonists, may also act asfunctional or receptor antagonists. Thus, IL-D80, IL-27, WSX-1/TCCR, orits antagonists, may be useful in the treatment of abnormal medicalconditions, including immune disorders, e.g., T cell immunedeficiencies, inflammation, or tissue rejection, or in cardiovascular orneurophysiological conditions.

[0040] The natural antigens are capable of mediating various biochemicalresponses which lead to biological or physiological responses in targetcells. The preferred embodiment characterized herein is from human, butother primate, or other species counterparts exist in nature. Additionalsequences for proteins in other mammalian species, e.g., primates,canines, felines, and rodents, should also be available, particularlythe domestic animal species. See below. The descriptions below aredirected, for exemplary purposes, to a human IL-D80 or IL-27, but arelikewise applicable to related embodiments from other species.

[0041] II. Purified IL-D80 or IL-27

[0042] Mammlian IL-D80 amino acid sequence, is shown in severalembodiments, e.g., SEQ ID NO: 2, 4, 6, or 8. EBI3 amino acid sequence isprovided in SEQ ID NO: 10. Other naturally occurring nucleic acids whichencode the protein can be isolated by standard procedures using theprovided sequence, e.g., PCR techniques, or by hybridization. Theseamino acid sequences, provided amino to carboxy, are important inproviding sequence information for the cytokine allowing fordistinguishing the protein antigen from other proteins and exemplifyingnumerous variants. Moreover, the peptide sequences allow preparation ofpeptides to generate antibodies to recognize such segments, andnucleotide sequences allow preparation of oligonucleotide probes, bothof which are strategies for detection or isolation, e.g., cloning, ofgenes encoding such sequences.

[0043] As used herein, the term “human soluble IL-D80 or IL-27” shallencompass, when used in a protein context, a protein having amino acidsequence corresponding to a soluble polypeptide shown in SEQ ID NO: 2 or6, or significant fragments thereof. Preferred embodiments comprise aplurality of distinct, e.g., nonoverlapping, segments of the specifiedlength. Typically, the plurality will be at least two, more usually atleast three, and preferably 5, 7, or even more. While the length minimaare provided, longer lengths, of various sizes, may be appropriate,e.g., one of length 7, and two of length 12.

[0044] Binding components, e.g., antibodies, typically bind to an IL-D80or IL-27 with high affinity, e.g., at least about 100 nM, usually betterthan about 30 nM, preferably better than about 10 nM, and morepreferably at better than about 3 nM. Counterpart proteins will be foundin mammalian species other than human, e.g., other primates, ungulates,or rodents. Non-mammalian species should also possess structurally orfunctionally related genes and proteins, e.g., birds or amphibians.

[0045] The term “polypeptide” as used herein includes a significantfragment or segment, and encompasses a stretch of amino acid residues ofat least about 8 amino acids, generally at least about 12 amino acids,typically at least about 16 amino acids, preferably at least about 20amino acids, and, in particularly preferred embodiments, at least about30 or more amino acids, e.g., 35, 40, 45, 50, 60, 75, 100, etc. Suchfragments may have ends which begin and/or end at virtually allpositions, e.g., beginning at residues 1, 2, 3, etc., and ending at,e.g., 150, 149, 148, etc., in all practical combinations. Particularlyinteresting peptides have ends corresponding to structural domainboundaries, e.g., helices A, B, C, and/or D.

[0046] The term “binding composition” refers to molecules that bind withspecificity to IL-D80 or IL-27, e.g., in an antibody-antigeninteraction. The specificity may be more or less inclusive, e.g.,specific to a particular embodiment, or to groups of relatedembodiments, e.g., primate, rodent, etc. It also includes compounds,e.g., proteins, which specifically associate with IL-D80 or IL-27,including in a natural physiologically relevant protein-proteininteraction, either covalent or non-covalent. The molecule may be apolymer, or chemical reagent. A functional analog may be a protein withstructural modifications, or it may be a molecule which has a molecularshape which interacts with the appropriate binding determinants. Thecompounds may serve as agonists or antagonists of a receptor bindinginteraction, see, e.g., Goodman, et al. (eds.) Goodman & Gilman's: ThePharmacological Bases of Therapeutics (current ed.) Pergamon Press.

[0047] Substantially pure, e.g., in a protein context, typically meansthat the protein is free from other contaminating proteins, nucleicacids, or other biologicals derived from the original source organism.Purity may be assayed by standard methods, typically by weight, and willordinarily be at least about 40% pure, generally at least about 50%pure, often at least about 60% pure, typically at least about 80% pure,preferably at least about 90% pure, and in most preferred embodiments,at least about 95% pure. Carriers or excipients will often be added.

[0048] Solubility of a polypeptide or fragment depends upon theenvironment and the polypeptide. Many parameters affect polypeptidesolubility, including temperature, electrolyte environment, size andmolecular characteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

[0049] The size and structure of the polypeptide should generally be ina substantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents.

[0050] The solvent and electrolytes will usually be a biologicallycompatible buffer, of a type used for preservation of biologicalactivities, and will usually approximate a physiological aqueoussolvent. Usually the solvent will have a neutral pH, typically betweenabout 5 and 10, and preferably about 7.5. On some occasions, one or moredetergents will be added, typically a mild non-denaturing one, e.g., CHS(cholesteryl hemisuccinate) or CHAPS(3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), or a lowenough concentration as to avoid significant disruption of structural orphysiological properties of the protein. In other instances, a harshdetergent may be used to effect significant denaturation.

[0051] The above will also be applicable to the IL-D80 or IL-27/EBI3composite cytokine, where SEQ ID NO: 10 is the polypeptide sequence ofEBI3.

[0052] III. Physical Variants

[0053] This invention also encompasses proteins or peptides havingsubstantial amino acid sequence identity with the amino acid sequence ofthe IL-D80 or IL-27 antigen. The variants include species, polymorphic,or allelic variants.

[0054] Amino acid sequence homology, or sequence identity, is determinedby optimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. The conservation may apply tobiological features, functional features, or structural features.Homologous amino acid sequences are typically intended to includenatural polymorphic or allelic and interspecies variations of a proteinsequence. Typical homologous proteins or peptides will have from 25-100%identity (if gaps can be introduced), to 50-100% identity (ifconservative substitutions are included) with the amino acid sequence ofthe IL-D80 or IL-27. Identity measures will be at least about 35%,generally at least about 40%, often at least about 50%, typically atleast about 60%, usually at least about 70%, preferably at least about80%, and more preferably at least about 90%.

[0055] The isolated IL-D80 or IL-27 DNA can be readily modified bynucleotide substitutions, nucleotide deletions, nucleotide insertions,and inversions of short nucleotide stretches. These modifications resultin novel DNA sequences which encode these antigens, their derivatives,or proteins having similar physiological, immunogenic, antigenic, orother functional activity. These modified sequences can be used toproduce mutant antigens or to enhance expression. Enhanced expressionmay involve gene amplification, increased transcription, increasedtranslation, and other mechanisms. “Mutant IL-D80 or IL-27” encompassesa polypeptide otherwise falling within the sequence identity definitionof the IL-D80 or IL-27 as set forth above, but having an amino acidsequence which differs from that of

[0056] IL-D80 or IL-27 as normally found in nature, whether by way ofdeletion, substitution, or insertion. This generally includes proteinshaving significant identity with a protein having sequence of SEQ ID NO:2, 4, 6, or 8, or the foregoing in association with SEQ ID NO: 10 and assharing various biological activities, e.g., antigenic or immunogenic,with those sequences, and in preferred embodiments contain most of thenatural full length disclosed sequences. Full length sequences willtypically be preferred, though truncated versions will also be useful,likewise, genes or proteins found from natural sources are typicallymost desired. Similar concepts apply to different IL-D80 or IL-27proteins, particularly those found in various warm blooded animals,e.g., mammals and birds. These descriptions are generally meant toencompass many IL-D80 or IL-27 proteins, not limited to the particularmammalian embodiments specifically discussed.

[0057] IL-D80 or IL-27 mutagenesis can also be conducted by making aminoacid insertions or deletions. Substitutions, deletions, insertions, orany combinations may be generated to arrive at a final construct.Insertions include amino- or carboxy-terminal fusions. Randommutagenesis can be conducted at a target codon and the expressed mutantscan then be screened for the desired activity. Methods for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known in the art, e.g., by M13 primer mutagenesis orpolymerase chain reaction (PCR) techniques. See, e.g., Sambrook, et al.(1989); Ausubel, et al. (1987 and Supplements); and Kunkel, et al.(1987) Methods in Enzymol. 154:367-382. Preferred embodiments include,e.g., 1-fold, 2-fold, 3-fold, 5-fold, 7-fold, etc., preferablyconservative substitutions at the nucleotide or amino acid levels.Preferably the substitutions will be away from the conserved cysteines,and often will be in the regions away from the helical structuraldomains. Such variants may be useful to produce specific antibodies, andoften will share many or all biological properties.

[0058] The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences.

[0059] In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.

[0060] The phosphoramidite method described by Beaucage and Carruthers(1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

[0061] Structural analysis can be applied to this gene, in comparison tothe IL-12 family of cytokines. In particular, β-sheet and α-helixresidues can be determined using, e.g., RASMOL program, see Bazan, etal. (1996) Nature 379:591; Lodi, et al. (1994) Science 263:1762-1766;Sayle and Milner-White (1995) TIBS 20:374-376; and Gronenberg, et al.(1991) Protein Engineering 4:263-269. Preferred residues forsubstitutions include the surface exposed residues which would bepredicted to interact with receptor. Other residues which shouldconserve function will be conservative substitutions, particularly atposition far from the surface exposed residues.

[0062] The above will also be applicable for the IL-D80 or IL-27 (i.e.,IL-D80+EBI3) composite cytokine where SEQ ID NO: 10 is the polypeptidesequence of EBI3.

[0063] IV. Functional Variants

[0064] The blocking of physiological response to IL-D80 or the

[0065] IL-27 composite cytokine may result from the competitiveinhibition of binding of the ligand to its receptor.

[0066] In vitro assays of the present invention will often use isolatedprotein, soluble fragments comprising receptor binding segments of theseproteins, or fragments attached to solid phase substrates. These assayswill also allow for the diagnostic determination of the effects ofeither binding segment mutations and modifications, or cytokinemutations and modifications, e.g., IL-D80 or IL-27 analogs.

[0067] This invention also contemplates the use of competitive drugscreening assays, e.g., where neutralizing antibodies to the cytokine,or receptor binding fragments compete with a test compound.

[0068] “Derivatives” of IL-D80 or IL-27 antigens include amino acidsequence mutants from naturally occurring forms, glycosylation variants,and covalent or aggregate conjugates with other chemical moieties.Covalent derivatives can be prepared by linkage of functionalities togroups which are found in IL-D80 or IL-27 amino acid side chains or atthe N- or C-termini, e.g., by standard means. See, e.g., Lundblad andNoyes (1988) Chemical Reagents for Protein Modification, vols. 1-2, CRCPress, Inc., Boca Raton, Fla.; Hugli (ed. 1989) Techniques in ProteinChemistry, Academic Press, San Diego, Calif.; and Wong (1991) Chemistryof Protein Conjugation and Cross Linking, CRC Press, Boca Raton, Fla.

[0069] In particular, glycosylation alterations are included, e.g., madeby modifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0070] Fusion polypeptides between IL-D80 or IL-27 and other homologousor heterologous proteins are also provided. Many cytokine receptors orother surface proteins are multimeric, e.g., homodimeric entities, and arepeat construct may have various advantages, including lessenedsusceptibility to proteolytic cleavage. Typical examples are fusions ofa reporter polypeptide, e.g., luciferase, with a segment or domain of aprotein, e.g., a receptor-binding segment, so that the presence orlocation of the fused ligand may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609. Other gene fusion partners includebacterial β-galactosidase, trpE, Protein A, B-lactamase, alpha amylase,alcohol dehydrogenase, yeast alpha mating factor, and detection orpurification tags such as a FLAG sequence of His6 sequence. See, e.g.,Godowski, et al. (1988) Science 241:812-816.

[0071] Fusion peptides will typically be made by either recombinantnucleic acid methods or by synthetic polypeptide methods. Techniques fornucleic acid manipulation and expression are described generally, e.g.,in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2ded.), vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al.(eds. 1993) Current Protocols in Molecular Biology, Greene and Wiley,NY. Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341-347; Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W.H. Freeman, NY. Refolding methodsmay be applicable to synthetic proteins.

[0072] This invention also contemplates the use of derivatives of IL-D80or IL-27 proteins other than variations in amino acid sequence orglycosylation. Such derivatives may involve covalent or aggregativeassociation with chemical moieties or protein carriers. Covalent oraggregative derivatives will be useful as immunogens, as reagents inimmunoassays, or in purification methods such as for affinitypurification of binding partners, e.g., other antigens. An IL-D80 orIL-27 can be immobilized by covalent bonding to a solid support such ascyanogen bromide-activated SEPHAROSE, by methods which are well known inthe art, or adsorbed onto polyolefin surfaces, with or withoutglutaraldehyde cross-linking, for use in the assay or purification ofanti-IL-D80 or IL-27 antibodies or an alternative binding composition.The IL-D80 or IL-27 proteins can also be labeled with a detectablegroup, e.g., for use in diagnostic assays. Purification of IL-D80 orIL-27 may be effected by an immobilized antibody or complementarybinding partner, e.g., binding portion of a receptor.

[0073] A solubilized IL-D80 or IL-27, or fragments of this invention canbe used as an immunogen for the production of antisera or antibodiesspecific for binding. Purified antigen can be used to screen monoclonalantibodies or antigen-binding fragments, encompassing antigen bindingfragments of natural antibodies, e.g., Fab, Fab′, F(ab)₂, etc. PurifiedIL-D80 or IL-27 antigens can also be used as a reagent to detectantibodies generated in response to the presence of elevated levels ofthe cytokine, which may be diagnostic of an abnormal or specificphysiological or disease condition. This invention contemplatesantibodies raised against amino acid sequences encoded by nucleotidesequence shown in SEQ ID NO: 1, 3, 5, or 7, or fragments of proteinscontaining it. Also contemplated are sequences encoding the IL-D80 orIL-27 cytokines, or fragments thereof. In particular, this inventioncontemplates antibodies having binding affinity to or being raisedagainst specific domains, e.g., helices A, B, C, or D.

[0074] The present invention contemplates the isolation of additionalclosely related species variants. Southern and Northern blot analysiswill establish that similar genetic entities exist in other mammals. Itis likely that IL-D80 or IL-27s are widespread in species variants,e.g., rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, andprimates.

[0075] The invention also provides means to isolate a group of relatedantigens displaying both distinctness and similarities in structure,expression, and function. Elucidation of many of the physiologicaleffects of the molecules will be greatly accelerated by the isolationand characterization of additional distinct species or polymorphicvariants of them. In particular, the present invention provides usefulprobes for identifying additional homologous genetic entities indifferent species.

[0076] The isolated genes will allow transformation of cells lackingexpression of an IL-D80 or IL-27, e.g., either species types or cellswhich lack corresponding proteins and exhibit negative backgroundactivity. This should allow analysis of the function of IL-D80 or IL-27in comparison to untransformed control cells.

[0077] Dissection of critical structural elements which effect thevarious physiological functions mediated through these antigens ispossible using standard techniques of modern molecular biology,particularly in comparing members of the related class. See, e.g., thehomolog-scanning mutagenesis technique described in Cunningham, et al.(1989) Science 243:1339-1336; and approaches used in O'Dowd, et al.(1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et al. (1990)EMBO J. 9:4381-4390.

[0078] Intracellular functions would probably involve receptorsignaling. However, protein internalization may occur under certaincircumstances, and interaction between intracellular components andcytokine may occur. Specific segments of interaction of IL-D80 or IL-27with interacting components may be identified by mutagenesis or directbiochemical means, e.g., cross-linking or affinity methods. Structuralanalysis by crystallographic or other physical methods will also beapplicable. Further investigation of the mechanism of signaltransduction will include study of associated components which may beisolatable by affinity methods or by genetic means, e.g.,complementation analysis of mutants.

[0079] Further study of the expression and control of IL-D80 or IL-27will be pursued. The controlling elements associated with the antigensshould exhibit differential physiological, developmental, tissuespecific, or other expression patterns. Upstream or downstream geneticregions, e.g., control elements, are of interest.

[0080] Structural studies of the IL-D80 or IL-27 antigens will lead todesign of new antigens, particularly analogs exhibiting agonist orantagonist properties on the molecule. This can be combined withpreviously described screening methods to isolate antigens exhibitingdesired spectra of activities.

[0081] V. Antibodies

[0082] Antibodies can be raised to various epitopes of the IL-D80 orIL-27 proteins, including species, polymorphic, or allelic variants, andfragments thereof, both in their naturally occurring forms and in theirrecombinant forms. Additionally, antibodies can be raised to IL-D80 orIL-27s in either their active forms or in their inactive forms,including native or denatured versions. Anti-idiotypic antibodies arealso contemplated.

[0083] Antibodies, including binding fragments and single chainversions, against predetermined fragments of the antigens can be raisedby immunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective IL-D80 or IL-27s, or screened foragonistic or antagonistic activity, e.g., mediated through a receptor.Antibodies may be agonistic or antagonistic, e.g., by stericallyblocking binding to a receptor. These monoclonal antibodies will usuallybind with at least a KD of about 1 mM, more usually at least about 300μM, typically at least about 100 μM, more typically at least about 30μM, preferably at least about 10 μM, and more preferably at least about3 μM or better.

[0084] An IL-D80 or IL-27 protein that specifically binds to or that isspecifically immunoreactive with an antibody generated against a definedimmunogen, such as an immunogen consisting of the amino acid sequence ofSEQ ID NO: 2, 4, 6, or 8, or any of the foregoing in association withSEQ ID NO: 10, is typically determined in an immunoassay. Theimmunoassay typically uses a polyclonal antiserum which was raised,e.g., to a polypeptide of SEQ ID NO: 2, 4, 6, or 8, or any of theforegoing in association with SEQ ID NO: 10. This antiserum is selectedto have low crossreactivity against other IL12 family members, e.g.,human or rodent IL-12, preferably from the same species, and any suchcrossreactivity is removed by immunoabsorption prior to use in theimmunoassay.

[0085] In order to produce antisera for use in an immunoassay, theprotein of SEQ ID NO: 2, 4, 6, or 8, or the foregoing in associationwith SEQ ID NO: 10, or a combination thereof, is isolated as describedherein. For example, recombinant protein may be produced in a mammaliancell line. An appropriate host, e.g., an inbred strain of mice such asBalb/c, is immunized with the selected protein, typically using astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see Harlow and Lane, supra). Alternatively, asynthetic peptide derived from the sequences disclosed herein andconjugated to a carrier protein can be used an immunogen. Polyclonalsera are collected and titered against the immunogen protein in animmunoassay, e.g., a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their cross reactivity againstother IL-12 family members, e.g., rodent IL-12, using a competitivebinding immunoassay such as the one described in Harlow and Lane, supra,at pages 570-573. Preferably at least one other IL-12 family member isused in this determination in conjunction with, e.g., the primate IL-12.The IL-12 family members can be produced as recombinant proteins andisolated using standard molecular biology and protein chemistrytechniques as described herein.

[0086] Immunoassays in the competitive binding format can be used forthe crossreactivity determinations. For example, the protein of SEQ IDNO: 2 or 6 can be immobilized to a solid support. Proteins added to theassay compete with the binding of the antisera to the immobilizedantigen. The ability of the above proteins to compete with the bindingof the antisera to the immobilized protein is compared to the protein ofSEQ ID NO: 2 or 6. Similarly, the composite cytokine of SEQ ID NO: 2 or6 in association with SEQ ID NO: 10 can be used. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the proteins listed above are selected and pooled. Thecross-reacting antibodies are then removed from the pooled antisera byimmunoabsorption with the above-listed proteins.

[0087] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein to the immunogen protein (e.g., the IL-12 like protein of SEQ IDNO: 2, 4, 6, or 8, or any of the foregoing in association with SEQ IDNO: 10). In order to make this comparison, the two proteins are eachassayed at a wide range of concentrations and the amount of each proteinrequired to inhibit 50% of the binding of the antisera to theimmobilized protein is determined. If the amount of the second proteinrequired is less than twice the amount of the protein of the selectedprotein or proteins that is required, then the second protein is said tospecifically bind to an antibody generated to the immunogen.

[0088] The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingto a receptor. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying IL-D80 or IL-27 protein or its receptors, e.g., WSX-1/TCCR(SEQ ID NO: 12). See, e.g., Chan (ed. 1987) Immunology: A PracticalGuide, Academic Press, Orlando, Fla.; Price and Newman (eds. 1991)Principles and Practice of Immunoassay, Stockton Press, N.Y.; and Ngo(ed. 1988) Nonisotopic Immunoassay, Plenum Press, N.Y. Crossabsorptions, depletions, or other means will provide preparations ofdefined selectivity, e.g., unique or shared species specificities. Thesemay be the basis for tests which will identify various groups ofantigens.

[0089] Further, the antibodies, including antigen binding fragments, ofthis invention can be potent antagonists that bind to the antigen andinhibit functional binding, e.g., to a receptor which may elicit abiological response. They also can be useful as non-neutralizingantibodies and can be coupled to toxins or radionuclides so that whenthe antibody binds to antigen, a cell expressing it, e.g., on itssurface, is killed. Further, these antibodies can be conjugated to drugsor other therapeutic agents, either directly or indirectly by means of alinker, and may effect drug targeting.

[0090] Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams, et al.(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane (1988) Antibodies: A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

[0091] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basicand Clinical Immunology (4th ed.), Lange Medical Publications, LosAltos, Calif., and references cited therein; Harlow and Lane (1988)Antibodies: A Laboratory Manual, CSH Press; Goding (1986) MonoclonalAntibodies: Principles and Practice (2d ed.), Academic Press, New York;and particularly in Kohler and Milstein (1975) in Nature 256:495-497,which discusses one method of generating monoclonal antibodies.

[0092] Other suitable techniques involve in vitro exposure oflymphocytes to the antigenic polypeptides or alternatively to selectionof libraries of antibodies in phage or similar vectors. See, Huse, etal. (1989) “Generation of a Large Combinatorial Library of theImmunoglobulin Repertoire in Phage Lambda,” Science 246:1275-1281; andWard, et al. (1989) Nature 341:544-546. The polypeptides and antibodiesof the present invention may be used with or without modification,including chimeric or humanized antibodies. Frequently, the polypeptidesand antibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

[0093] The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.(1984) Meth. Enzymol. 104:3-55. The converse may be used to purifyantibodies.

[0094] Antibodies raised against each IL-D80 or IL-27 will also beuseful to raise anti-idiotypic antibodies. These will be useful indetecting or diagnosing various immunological conditions related toexpression of the respective antigens.

[0095] VI. Nucleic Acids

[0096] The described peptide sequences and the related reagents areuseful in detecting, isolating, or identifying a DNA clone encodingIL-D80 or IL-27, e.g., from a natural source. Typically, it will beuseful in isolating a gene from mammal, and similar procedures will beapplied to isolate genes from other species, e.g., warm blooded animals,such as birds and mammals. Cross hybridization will allow isolation ofIL-D80 or IL-27 from the same, e.g., polymorphic variants, or otherspecies. A number of different approaches will be available tosuccessfully isolate a suitable nucleic acid clone.

[0097] The purified protein or defined peptides are useful forgenerating antibodies by standard methods, as described above. Syntheticpeptides or purified protein can be presented to an immune system togenerate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991)Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.

[0098] For example, the specific binding composition could be used forscreening of an expression library made from a cell line which expressesan IL-D80 or IL-27. Screening of intracellular expression can beperformed by various staining or immunofluorescence procedures. Bindingcompositions could be used to affinity purify or sort out cellsexpressing a surface fusion protein.

[0099] The peptide segments can also be used to predict appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., SEQ ID NO: 1, 3, 5, or 7, or any of the foregoing in addition toSEQ ID NO: 9. In combination with polymerase chain reaction (PCR)techniques, synthetic oligonucleotides will be useful in selectingcorrect clones from a library. Complementary sequences will also be usedas probes, primers, or antisense strands. Various fragments should beparticularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

[0100] This invention contemplates use of isolated DNA or fragments toencode an antigenic or biologically active corresponding IL-D80 or IL-27polypeptide, particularly lacking the portion coding the untranslated 5′portion of the described sequence. In addition, this invention coversisolated or recombinant DNA which encodes a biologically active proteinor polypeptide and which is capable of hybridizing under appropriateconditions with the DNA sequences described herein. Said biologicallyactive protein or polypeptide can be an intact antigen, or fragment, andhave an amino acid sequence disclosed in, e.g., SEQ ID NO: 2, 4, 6, or8, or any of the foregoing in association with SEQ ID NO: 10,particularly a mature, secreted polypeptide. Further, this inventioncovers the use of isolated or recombinant DNA, or fragments thereof,which encode proteins which exhibit high identity to a secreted IL-D80or IL-27. The isolated DNA can have the respective regulatory sequencesin the 5′ and 3′ flanks, e.g., promoters, enhancers, poly-A additionsignals, and others. Alternatively, expression may be effected byoperably linking a coding segment to a heterologous promoter, e.g., byinserting a promoter upstream from an endogenous gene.

[0101] An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA,or a mixed polymer, which is substantially separated from othercomponents which naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Generally, the nucleic acid will be in a vector or fragment less thanabout 50 kb, usually less than about 30 kb, typically less than about 10kb, and preferably less than about 6 kb.

[0102] An isolated nucleic acid will generally be a homogeneouscomposition of molecules, but will, in some embodiments, contain minorheterogeneity. This heterogeneity is typically found at the polymer endsor portions not critical to a desired biological function or activity.

[0103] A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

[0104] Alternatively, it is performed to join together nucleic acidsegments of desired functions to generate a single genetic entitycomprising a desired combination of functions not found in the commonlyavailable natural forms. Restriction enzyme recognition sites are oftenthe target of such artificial manipulations, but other site specifictargets, e.g., promoters, DNA replication sites, regulation sequences,control sequences, or other useful features may be incorporated bydesign. A similar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesor polymorphic variants.

[0105] A significant “fragment” in a nucleic acid context is acontiguous segment of at least about 17 nucleotides, generally at leastabout 22 nucleotides, ordinarily at least about 29 nucleotides, moreoften at least about 35 nucleotides, typically at least about 41nucleotides, usually at least about 47 nucleotides, preferably at leastabout 55 nucleotides, and in particularly preferred embodiments will beat least about 60 or more nucleotides, e.g., 67, 73, 81, 89, 95, 150,200, 250, 300, 500, etc.

[0106] A DNA which codes for an IL-D80 or IL-27 protein will beparticularly useful to identify genes, mRNA, and cDNA species which codefor related or similar proteins, as well as DNAs which code forhomologous proteins from different species. There will be homologs inother species, including primates, rodents, canines, felines, birds, andfish.

[0107] Various IL-D80 or IL-27 proteins should be homologous and areencompassed herein. However, even proteins that have a more distantevolutionary relationship to the antigen can readily be isolated underappropriate conditions using these sequences if they are sufficientlyhomologous. Primate IL-D80 or IL-27 proteins are of particular interest.

[0108] Recombinant clones derived from the genomic sequences, e.g.,containing introns, will be useful for transgenic studies, including,e.g., transgenic cells and organisms, and for gene therapy. See, e.g.,Goodnow (1992) “Transgenic Animals” in Roitt (ed.) Encyclopedia ofImmunology, Academic Press, San Diego, pp. 1502-1504; Travis (1992)Science 256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi(1989) Science 244:1288; Robertson (ed. 1987) Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, IRL Press, Oxford; Rosenberg(1992) J. Clinical Oncology 10:180-199; and Cournoyer and Caskey (1993)Ann. Rev. Immunol. 11:297-329. Alternatively, expression may be effectedby operably linking a coding segment to a heterologous promoter, e.g.,by inserting a promoter upstream from an endogenous gene. See, e.g.,Treco, et al. WO96/29411 or U.S. Ser. No. 08/406,030.

[0109] Substantial homology, e.g., identity, in the nucleic acidsequence comparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least about 58%,ordinarily at least about 65%, often at least about 71%, typically atleast about 77%, usually at least about 85%, preferably at least about95 to 98% or more, and in particular embodiments, as high as about 99%or more of the nucleotides. Alternatively, substantial homology existswhen the segments will hybridize under selective hybridizationconditions, to a strand, or its complement, typically using a sequenceof IL-D80 or IL-27, e.g., in SEQ ID NO: 1, 3, 5, or 7, or any of theforegoing in association with SEQ ID NO: 9. Typically, selectivehybridization will occur when there is at least about 55% identity overa stretch of at least about 30 nucleotides, preferably at least about75% over a stretch of about 25 nucleotides, and most preferably at leastabout 90% over about 20 nucleotides. See, Kanehisa (1984) Nuc. AcidsRes. 12:203-213. The length of identity comparison, as described, may beover longer stretches, and in certain embodiments will be over a stretchof at least about 17 nucleotides, usually at least about 28 nucleotides,typically at least about 40 nucleotides, and preferably at least about75 to 100 or more nucleotides.

[0110] Stringent conditions, in referring to homology in thehybridization context, will be stringent combined conditions of salt,temperature, organic solvents, and other parameters, typically thosecontrolled in hybridization reactions. Stringent temperature conditionswill usually include temperatures in excess of about 30° C., usually inexcess of about 37° C., typically in excess of about 55° C., 60° C., or65° C., and preferably in excess of about 70° C. Stringent saltconditions will ordinarily be less than about 1000 mM, usually less thanabout 400 mM, typically less than about 250 mM, preferably less thanabout 150 mM, including about 100, 50, or even 20 mM. However, thecombination of parameters is much more important than the measure of anysingle parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370. Hybridization under stringent conditions should give abackground of at least 2-fold over background, preferably at least 3-5or more.

[0111] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0112] Optical alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith and Waterman (1981) Adv.Appl. Math. 2:482, by the homology alignment algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methodof Pearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

[0113] One example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendrogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng and Doolittle(1987) J. Mol. Evol. 35:351-360. The method used is similar to themethod described by Higgins and Sharp (1989) CABIOS 5:151-153. Theprogram can align up to 300 sequences, each of a maximum length of 5,000nucleotides or amino acids. The multiple alignment procedure begins withthe pairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster is then aligned to thenext most related sequence or cluster of aligned sequences. Two clustersof sequences are aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

[0114] Another example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described Altschul, et al. (1990) J. Mol. Biol.215:403-410. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http:www.ncbi.nlm.nih.gov/). This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul, et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extension of the word hits in each direction are halted when:the cumulative alignment score falls off by the quantity X from itsmaximum achieved value; the cumulative score goes to zero or below, dueto the accumulation of one or more negative-scoring residue alignments;or the end of either sequence is reached. The BLAST algorithm parametersW, T, and X determine the sensitivity and speed of the alignment. TheBLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62scoring matrix (see Henikoff and Henikoff (1989) Proc. Nat'l Acad. Sci.USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=4, anda comparison of both strands.

[0115] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

[0116] A further indication that two nucleic acid sequences ofpolypeptides are substantially identical is that the polypeptide encodedby the first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

[0117] IL-D80 or IL-27 from other mammalian species can be cloned andisolated by cross-species hybridization of closely related species.Homology may be relatively low between distantly related species, andthus hybridization of relatively closely related species is advisable.Alternatively, preparation of an antibody preparation which exhibitsless species specificity may be useful in expression cloning approaches.

[0118] VII. Making IL-D80 or IL-27; Mimetics

[0119] DNA which encodes the IL-D80 or IL-27 or fragments thereof can beobtained by chemical synthesis, screening cDNA libraries, or screeninggenomic libraries prepared from a wide variety of cell lines or tissuesamples. See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170;Gubler and Hoffman (1983) Gene 25:263-269; and Glover (ed. 1984) DNACloning: A Practical Approach, IRL Press, Oxford. Alternatively, thesequences provided herein provide useful PCR primers or allow syntheticor other preparation of suitable genes encoding an IL-D80 or IL-27;including naturally occurring embodiments.

[0120] This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length IL-D80 or IL-27 or fragments which can inturn, e.g., be used to generate polyclonal or monoclonal antibodies; forbinding studies; for construction and expression of modified molecules;and for structure/function studies. There may be a need for a chaparoneprotein for efficient secretion, or additional steps may be necessary toretrieve the protein from the intracellular compartment.

[0121] Vectors, as used herein, comprise plasmids, viruses,bacteriophage, integratable DNA fragments, and other vehicles whichenable the integration of DNA fragments into the genome of the host.See, e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: ALaboratory Manual, Elsevier, N.Y.; and Rodriguez, et al. (eds. 1988)Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Buttersworth, Boston, Mass.

[0122] For purposes of this invention, DNA sequences are operably linkedwhen they are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See, e.g., Rodriguez, et al., Chapter 10, pp. 205-236;Balbas and Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel,et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley,NY.

[0123] Representative examples of suitable expression vectors includepCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142;pMCl1eo Poly-A, see Thomas, et al. (1987) Cell 51:503-512; and abaculovirus vector such as pAC 373 or pAC 610. See, e.g., Miller (1988)Ann. Rev. Microbiol. 42:177-199.

[0124] It will often be desired to express an IL-D80 or IL-27polypeptide in a system which provides a specific or definedglycosylation pattern. See, e.g., Luckow and Summers (1988)Bio/Technology 6:47-55; and Kaufman (1990) Meth. Enzymol. 185:487-511.

[0125] The IL-D80 or IL-27, or a fragment thereof, may be engineered tobe phosphatidyl inositol (PI) linked to a cell membrane, but can beremoved from membranes by treatment with a phosphatidyl inositolcleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. Thisreleases the antigen in a biologically active form, and allowspurification by standard procedures of protein chemistry. See, e.g., Low(1989) Biochim. Biophys. Acta 988:427-454; Tse, et al. (1985) Science230:1003-1008; and Brunner, et al. (1991) J. Cell Biol. 114:1275-1283.

[0126] Now that the IL-D80 or IL-27 has been characterized, fragments orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis, Springer-Verlag, New York; Bodanszky (1984) ThePrinciples of Peptide Synthesis, Springer-Verlag, New York; andVillafranca (ed. 1991) Techniques in Protein Chemistry II, AcademicPress, San Diego, Calif.

[0127] VIII. Uses

[0128] The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in IL-D80or IL-27 mediated conditions, or below in the description of kits fordiagnosis. The gene may be useful in forensic sciences, e.g., todistinguish rodent from human, or as a marker to distinguish betweendifferent cells exhibiting differential expression or modificationpatterns.

[0129] This invention also provides reagents with significant commercialand/or therapeutic potential. The IL-D80 or IL-27 (naturally occurringor recombinant), fragments thereof, and antibodies thereto, along withcompounds identified as having binding affinity to IL-D80 or IL-27,should be useful as reagents for teaching techniques of molecularbiology, immunology, or physiology. Appropriate kits may be preparedwith the reagents, e.g., in practical laboratory exercises in productionor use of proteins, antibodies, cloning methods, histology, etc.

[0130] The reagents will also be useful in the treatment of conditionsassociated with abnormal physiology or development, includinginflammatory conditions. They may be useful in vitro tests for presenceor absence of interacting components, which may correlate with successof particular treatment strategies. In particular, modulation ofphysiology of various, e.g., hematopoietic or lymphoid, cells will beachieved by appropriate methods for treatment using the compositionsprovided herein. See, e.g., Thomson (ed. 1998) The Cytokine Handbook (3ded.) Academic Press, San Diego; Metcalf and Nicola (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press; andAggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

[0131] For example, a disease or disorder associated with abnormalexpression or abnormal signaling by an IL-D80 or IL-27 should be alikely target for an agonist or antagonist. Similarly, the bindingpartner of the IL-27 composite cytokine, WSX-1/TCCR, should also be atarget. The new cytokine should play a role in regulation or developmentof hematopoietic cells, e.g., lymphoid cells, which affect immunologicalresponses, e.g., inflammation and/or autoimmune disorders.Alternatively, it may affect vascular physiology or development, orneuronal effects.

[0132] In particular, the cytokine should mediate, in various contexts,cytokine synthesis by the cells, proliferation, etc. Antagonists ofIL-D80 or IL-27, such as mutein variants of a naturally occurring formof IL-D80 or IL-27 or blocking antibodies, may provide a selective andpowerful way to block immune responses, e.g., in situations asinflammatory or autoimmune responses. See also Samter, et al. (eds.)Immunological Diseases vols. 1 and 2, Little, Brown and Co.

[0133] Various abnormal conditions are known in different cell typeswhich will produce IL-D80 or IL-27, e.g., as evaluated by mRNAexpression by Northern blot analysis. See Berkow (ed.) The Merck Manualof Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn, et al.Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.; andWeatherall, et al. (eds.) Oxford Textbook of Medicine, Oxford UniversityPress, Oxford. Many other medical conditions and diseases involveactivation by macrophages or monocytes, and many of these will beresponsive to treatment by an agonist or antagonist provided herein.See, e.g., Stites and Terr (eds.; 1991) Basic and Clinical ImmunologyAppleton and Lange, Norwalk, Conn.; and Samter, et al. (eds.)Immunological Diseases Little, Brown and Co. These problems should besusceptible to prevention or treatment using compositions providedherein.

[0134] IL-D80 or IL-27, antagonists, antibodies, etc., can be purifiedand then administered to a patient, veterinary or human. These reagentscan be combined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers, excipients, or preservatives. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies orbinding fragments thereof, including forms which are not complementbinding.

[0135] Drug screening using IL-D80, IL-27, WSX-1/TCCR or fragmentsthereof can be performed to identify compounds having binding affinityto or other relevant biological effects on IL-D80 or IL-27 functions,including isolation of associated components. Subsequent biologicalassays can then be utilized to determine if the compound has intrinsicstimulating activity and is therefore a blocker or antagonist in that itblocks the activity of the cytokine. Likewise, a compound havingintrinsic stimulating activity can activate the signal pathway and isthus an agonist in that it simulates the activity of IL-D80 or IL-27.This invention further contemplates the therapeutic use of blockingantibodies to IL-D80, IL-27, or WSX-1/TCCR as antagonists and ofstimulatory antibodies as agonists. This approach should be particularlyuseful with other IL-D80 or IL-27 species variants.

[0136] The quantities of reagents necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, latestEd., Pergamon Press; and Remington's Pharmaceutical Sciences, latested., Mack Publishing Co., Easton, Penn. Methods for administration arediscussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer (1990) Science249:1527-1533.

[0137] IL-D80 or IL-27, fragments thereof, and antibodies to it or itsfragments, antagonists, and agonists, may be administered directly tothe host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, topical, or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. See, e.g., Gilman, et al.(eds. 1990) Goodman and Gilman's: The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, etal. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York; Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Tablets, Dekker, New York; and Lieberman, et al. (eds. 1990)Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York. Thetherapy of this invention may be combined with or used in associationwith other agents, e.g., other cytokines, including IL-12, or itsantagonists.

[0138] Both naturally occurring and recombinant forms of the IL-D80 orIL-27s of this invention are particularly useful in kits and assaymethods which are capable of screening compounds for binding activity tothe proteins. Several methods of automating assays have been developedin recent years so as to permit screening of tens of thousands ofcompounds in a short period. See, e.g., Fodor, et al. (1991) Science251:767-773, which describes means for testing of binding affinity by aplurality of defined polymers synthesized on a solid substrate. Thedevelopment of suitable assays can be greatly facilitated by theavailability of large amounts of purified, soluble IL-D80 or IL-27 asprovided by this invention.

[0139] Other methods can be used to determine the critical residues inIL-D80 or IL-27 receptor interactions. Mutational analysis can beperformed, e.g., see Somoza, et al. (1993) J. Exptl. Med. 178:549-558,to determine specific residues critical in the interaction and/orsignaling. PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (Kingand Sternberg (1996) Protein Sci. 5:2298-2310) can provide secondarystructure predictions of α-helix (H), β-strand (E), or coil (L). HelicesA and D are most important in receptor interaction, with the D helix themore important region. Boundaries for the various helices are indicatedabove. Surface exposed residues would affect receptor binding, whileembedded residues would affect general structure.

[0140] For example, antagonists can normally be found once the antigenhas been structurally defined, e.g., by tertiary structure data. Testingof potential interacting analogs is now possible upon the development ofhighly automated assay methods using a purified IL-D80 or IL-27. Inparticular, new agonists and antagonists will be discovered by usingscreening techniques described herein. Of particular importance arecompounds found to have a combined binding affinity for a spectrum ofIL-D80 or IL-27 molecules, e.g., compounds which can serve asantagonists for species variants of IL-D80 or IL-27.

[0141] One method of drug screening utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant DNA moleculesexpressing an IL-D80 or IL-27. Cells may be isolated which express anIL-D80 or IL-27 in isolation from other molecules. Such cells, either inviable or fixed form, can be used for standard binding partner bindingassays. See also, Parce, et al. (1989) Science 246:243-247; and Owicki,et al. (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describesensitive methods to detect cellular responses.

[0142] Another technique for drug screening involves an approach whichprovides high throughput screening for compounds having suitable bindingaffinity to an IL-D80 or IL-27 and is described in detail in Geysen,European Patent Application 84/03564, published on Sep. 13, 1984. First,large numbers of different small peptide test compounds are synthesizedon a solid substrate, e.g., plastic pins or some other appropriatesurface, see Fodor, et al. (1991). Then all the pins are reacted withsolubilized, unpurified or solubilized, purified IL-D80 or IL-27, andwashed. The next step involves detecting bound IL-D80 or IL-27.

[0143] Rational drug design may also be based upon structural studies ofthe molecular shapes of the IL-D80 or IL-27 and other effectors oranalogs. Effectors may be other proteins which mediate other functionsin response to binding, or other proteins which normally interact withIL-D80 or IL-27, e.g., a receptor. One means for determining which sitesinteract with specific other proteins is a physical structuredetermination, e.g., x-ray crystallography or 2 dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions, as modeled, e.g., against othercytokine-receptor models. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York.

[0144] IX. Kits

[0145] This invention also contemplates use of IL-D80 or IL-27 proteins,fragments thereof, peptides, and their fusion products in a variety ofdiagnostic kits and methods for detecting the presence of another IL-D80or IL-27 or binding partner. Typically the kit will have a compartmentcontaining either a defined IL-D80 or IL-27 peptide or gene segment or areagent which recognizes one or the other, e.g., IL-D80 or IL-27fragments or antibodies.

[0146] A kit for determining the binding affinity of a test compound toan IL-D80 or IL-27 would typically comprise a test compound; a labeledcompound, for example a binding partner or antibody having known bindingaffinity for IL-D80 or IL-27; a source of IL-D80 or IL-27 (naturallyoccurring or recombinant); and a means for separating bound from freelabeled compound, such as a solid phase for immobilizing the molecule.Once compounds are screened, those having suitable binding affinity tothe antigen can be evaluated in suitable biological assays, as are wellknown in the art, to determine whether they act as agonists orantagonists to the IL-D80 or IL-27 signaling pathway. The availabilityof recombinant IL-D80 or IL-27 polypeptides also provide well definedstandards for calibrating such assays.

[0147] A preferred kit for determining the concentration of, e.g., anIL-D80 or IL-27 in a sample would typically comprise a labeled compound,e.g., binding partner or antibody, having known binding affinity for theantigen, a source of cytokine (naturally occurring or recombinant) and ameans for separating the bound from free labeled compound, e.g., a solidphase for immobilizing the IL-D80 or IL-27. Compartments containingreagents, and instructions, will normally be provided.

[0148] Antibodies, including antigen binding fragments, specific for theIL-D80 or IL-27 or fragments are useful in diagnostic applications todetect the presence of elevated levels of IL-D80 or IL-27 and/or itsfragments. Such diagnostic assays can employ lysates, live cells, fixedcells, immunofluorescence, cell cultures, body fluids, and further caninvolve the detection of antigens related to the antigen in serum, orthe like. Diagnostic assays may be homogeneous (without a separationstep between free reagent and antigen-binding partner complex) orheterogeneous (with a separation step). Various commercial assays exist,such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassaytechnique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), andthe like. See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525;Harlow and Lane (1980) Antibodies: A Laboratory Manual, CSH Press, NY;and Coligan, et al. (eds. 1993) Current Protocols in Immunology, Greeneand Wiley, NY.

[0149] Anti-idiotypic antibodies may have similar use to diagnosepresence of antibodies against an IL-D80 or IL-27, as such may bediagnostic of various abnormal states. For example, overproduction ofIL-D80 or IL-27 may result in production of various immunologicalreactions which may be diagnostic of abnormal physiological states,particularly in proliferative cell conditions such as cancer or abnormalactivation or differentiation. Moreover, the distribution patternavailable provides information that the cytokine is expressed inpancreatic islets, suggesting the possibility that the cytokine may beinvolved in function of that organ, e.g., in a diabetes relevant medicalcondition.

[0150] Frequently, the reagents for diagnostic assays are supplied inkits, so as to optimize the sensitivity of the assay. For the subjectinvention, depending upon the nature of the assay, the protocol, and thelabel, either labeled or unlabeled antibody or binding partner, orlabeled IL-D80 or IL-27 is provided. This is usually in conjunction withother additives, such as buffers, stabilizers, materials necessary forsignal production such as substrates for enzymes, and the like.Preferably, the kit will also contain instructions for proper use anddisposal of the contents after use. Typically the kit has compartmentsfor each useful reagent. Desirably, the reagents are provided as a drylyophilized powder, where the reagents may be reconstituted in anaqueous medium providing appropriate concentrations of reagents forperforming the assay.

[0151] Many of the aforementioned constituents of the drug screening andthe diagnostic assays may be used without modification or may bemodified in a variety of ways. For example, labeling may be achieved bycovalently or non-covalently joining a moiety which directly orindirectly provides a detectable signal. In any of these assays, thebinding partner, test compound, IL-D80 or IL-27, or antibodies theretocan be labeled either directly or indirectly. Possibilities for directlabeling include label groups: radiolabels such as ¹²⁵I, enzymes (U.S.Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, andfluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring thechange in fluorescence intensity, wavelength shift, or fluorescencepolarization. Possibilities for indirect labeling include biotinylationof one constituent followed by binding to avidin coupled to one of theabove label groups.

[0152] There are also numerous methods of separating the bound from thefree IL-D80 or IL-27, or alternatively the bound from the free testcompound. The IL-D80 or IL-27 can be immobilized on various matrixesfollowed by washing. Suitable matrixes include plastic such as an ELISAplate, filters, and beads. See, e.g., Coligan, et al. (eds. 1993)Current Protocols in Immunology, Vol. 1, Chapter 2, Greene and Wiley,NY. Other suitable separation techniques include, without limitation,the fluorescein antibody magnetizable particle method described inRattle, et al. (1984) Clin. Chem. 30:1457-1461, and the double antibodymagnetic particle separation as described in U.S. Pat. No. 4,659,678.

[0153] Methods for linking proteins or their fragments to the variouslabels have been extensively reported in the literature and do notrequire detailed discussion here. Many of the techniques involve the useof activated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

[0154] Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan IL-D80 or IL-27. These sequences can be used as probes for detectinglevels of the IL-D80 or IL-27 message in samples from patients suspectedof having an abnormal condition, e.g., inflammatory or autoimmune. Sincethe cytokine may be a marker or mediator for activation, it may beuseful to determine the numbers of activated cells to determine, e.g.,when additional therapy may be called for, e.g., in a preventativefashion before the effects become and progress to significance. Thepreparation of both RNA and DNA nucleotide sequences, the labeling ofthe sequences, and the preferred size of the sequences has receivedample description and discussion in the literature. See, e.g.,Langer-Safer, et al. (1982) Proc. Nat'l. Acad. Sci. 79:4381-4385; Caskey(1987) Science 236:962-967; and Wilchek et al. (1988) Anal. Biochem.171:1-32.

[0155] Diagnostic kits which also test for the qualitative orquantitative expression of other molecules are also contemplated.Diagnosis or prognosis may depend on the combination of multipleindications used as markers. Thus, kits may test for combinations ofmarkers. See, e.g., Viallet, et al. (1989) Progress in Growth FactorRes. 1:89-97. Other kits may be used to evaluate other cell subsets.

[0156] X. Isolating an IL-D80 or IL-27 Receptor

[0157] Having isolated a ligand of a specific ligand-receptorinteraction, methods exist for isolating the receptor. See, Gearing, etal. (1989) EMBO J. 8:3667-3676. For example, means to label the IL-D80or IL-27 cytokine without interfering with the binding to its receptorcan be determined. For example, an affinity label can be fused to eitherthe amino- or carboxyl-terminus of the ligand. Such label may be a FLAGepitope tag, or, e.g., an Ig or Fc domain. An expression library can bescreened for specific binding of the cytokine, e.g., by cell sorting, orother screening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA90:11267-11271; and Liu, et al. (1994) J. Immunol. 152:1821-29.Alternatively, a panning method may be used. See, e.g., Seed and Aruffo(1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369.

[0158] Protein cross-linking techniques with label can be applied toisolate binding partners of the IL-D80 or IL-27 cytokine. This wouldallow identification of proteins which specifically interact with thecytokine, e.g., in a ligand-receptor like manner. It has been shown, asnoted below, that the IL-27 composite cytokine binds at least to anIL-12R-like subunit known as WSX-1/TCCR.

[0159] FACS analysis of detectably stained IL-D80, EBI3, and WSX-1/TCCRmolecules led to the finding that these molecules are components in areceptor subunit/ligand complex. Specifically, the composite cytokine ofE-tagged hIL-D80 (hIL-D80E) and F-tagged (Flag-tagged) hEBI3 (FhEBI3)binds to Baf3 cells expressing an F-tagged version of WSX-1/TCCR, alsoreferred to as hNR30. The cells were stained using anti-E mAb and aPE-conjugated anti-mouse Fab₂ fragment. Co-immunoprecipitationexperiments also indicated that hIL-27 could be immunoprecipitated withR-tagged (RGSH₆-tagged) soluble WSX-1/TCCR (shNR30R). Alternatively,shNR30R could be co-immunoprecipated in the presence of hIL-D80E/FhEBI3complex using anti-E or anti-F mAbs. These experiments establish thatWSX-1/TCCR is a receptor component of the IL-27 composite cytokine.Recent evidence shows that disrupting the WSX-1/TCCR gene in miceresults in lowered expression of IFNγ, which is a critical cytokine inthe mediation of pro-inflammatory functions. These mice were unable tomount a Th1 response (See, e.g., Chen, et al. (2000) Nature407:916-920.)

[0160] Experimental data indicates a possible role for the IL-27composite cytokine in driving an inflammatory response. The expressionprofile of EBI3 and IL-D80 overlaps in monocytes, macrophages, anddendritic cells, indicating that the composite cytokine is primarilyproduced by antigent presenting cells (APCs) of the immune system. EBI3has been shown to be upregulated in colonic tissue of patients sufferingfrom gut inflammation disorders, e.g., ulcerative colitis, suggestingthat the composite cytokine may also be involved.

[0161] Taken together the above indicates a role for the compositecytokine and its associated receptor subunit WSX-1/TCCR in inflammatoryresponses. Therefore antagonizing the function of any of the componentsin the receptor subunit:ligand complex should have a beneficial effectin inflammatory diseases, e.g., inflammatory bowel disease, rheumatoidarthritis, etc.

EXAMPLES

[0162] I. General Methods

[0163] Many of the standard methods below are described or referenced,e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory ManualCold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook,et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3,CSH Press, NY; Ausubel, et al., Biology Greene Publishing Associates,Brooklyn, NY; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology Wiley/Greene, NY; Innis, et al. (eds.1990) PCR Protocols: A Guide to Methods and Applications Academic Press,NY. Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; Coligan, et al. (1995 and supplements) Current Protocols inProtein Science John Wiley and Sons, New York, N.Y.; P. Matsudaira (ed.1993) A Practical Guide to Protein and Peptide Purification forMicrosequencing, Academic Press, San Diego, Calif.; and manufacturer'sliterature on use of protein purification products, e.g., Pharmacia,Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination withrecombinant techniques allow fusion to appropriate segments (epitopetags), e.g., to a FLAG sequence or an equivalent which can be fused,e.g., via a protease-removable sequence. See, e.g., Hochuli (1989)Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, NY;and Crowe, et al. (1992) QIAexpress: The High Level Expression & ProteinPurification System QUIAGEN, Inc., Chatsworth, Calif.

[0164] Standard immunological techniques are described, e.g., inHertzenberg, et al. (eds. 1996) Weir's Handbook of ExperimentalImmunology vols. 1-4, Blackwell Science; Coligan (1991) CurrentProtocols in Immunology Wiley/Greene, NY; and Methods in Enzymologyvols. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.Cytokine assays are described, e.g., in Thomson (ed. 1998) The CytokineHandbook (3d ed.) Academic Press, San Diego; Mire-Sluis and Thorpe(1998) Cytokines Academic Press, San Diego; Metcalf and Nicola (1995)The Hematopoietic Colony Stimulating Factors Cambridge University Press;and Aggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

[0165] Assays for vascular biological activities are well known in theart. They will cover angiogenic and angiostatic activities in tumor, orother tissues, e.g., arterial smooth muscle proliferation (see, e.g.,Koyoma, et al. (1996) Cell 87:1069-1078), monocyte adhesion to vascularepithelium (see McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc.See also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am.J. Pathol. 147:668-677; Thyberg, et al. (1990) Atherosclerosis10:966-990; and Gumbiner (1996) Cell 84:345-357.

[0166] Assays for neural cell biological activities are described, e.g.,in Wouterlood (ed. 1995) Neuroscience Protocols modules 10, Elsevier;Methods in Neurosciences Academic Press; and Neuromethods Humana Press,Totowa, N.J. Methodology of developmental systems is described, e.g., inMeisami (ed.) Handbook of Human Growth and Developmental Biology CRCPress; and Chrispeels (ed.) Molecular Techniques and Approaches inDevelopmental Biology Interscience.

[0167] FACS analyses are described in Melamed, et al. (1990) FlowCytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al.(1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.

[0168] II. Cloning of Human IL-D80

[0169] The sequences of primate, e.g., human, genes are provided in SEQID NO: 1, 3, 5, and 7. These sequences are derived from a sequencedatabase. These sequences allow preparation of PCR primers, or probes,to determine cellular distribution of the gene. These sequences allowisolation of genomic DNA which encode the message.

[0170] Using the probe or PCR primers, various tissues or cell types areprobed to determine cellular distribution. PCR products are clonedusing, e.g., a TA cloning kit (Invitrogen). The resulting cDNA plasmidsare sequenced from both termini on an automated sequencer (AppliedBiosystems).

[0171] A structural alignment of available IL-6 family cytokine folds(CNTF, LIF, IL-6, OSM and GCSF) from FSSP (see, e.g., Holm and Sander(1998) Nucleic Acids Res. 26:316-319 was profile-aligned to othersequences (including distant species variants of the aforementionedcytokines, plus CT-1, GPA and viral IL-6's) with Clustal X (see, e.g.,Thompson, et al. (1997) Nucleic Acids Res. 25:4876-4882) with somemanual adjustment. A weighted profile (see, e.g., Thompson, et al.(1994) Nucleic Acids Res. 22:4673-4680) of the most conserved region ofthe fold, the C-terminal D-helix segment, a ˜40 amino acid block, wascreated. Fast scans of sequence databases on a Bioccelerator machine(Compugen, Tel Aviv, Israel) with the Profilesearch program (Gribskov etal., 1987) identified human EST A1085007, mouse EST AA266872 andeventually, the identification of a novel hemopoietic cytokine. Thecytokine was initially referred to as IL-D80, but is also known as p28according to its apparent molecular mass as determined by SDS-PAGE.

[0172] III. Cellular Expression of IL-D8 or IL-27

[0173] An appropriate probe or primers specific for cDNA encodingprimate IL-D80 or IL-27 are prepared. Typically, the probe is labeled,e.g., by random priming.

[0174] Southern Analysis: DNA (5 μg) from a primary amplified cDNAlibrary was digested with appropriate restriction enzymes to release theinserts, run on a 1% agarose gel and transferred to a nylon membrane(Schleicher and Schuell, Keene, N.H.).

[0175] Samples for human mRNA isolation may include: peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T109); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); Tcell tumor lines Jurkat and Hut78, resting (T117); T cell clones, pooledAD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118); T cellrandom γδ T cell clones, resting (T119); CD28-T cell clone; Splenocytes,resting (B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); Bcell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting(B102); B cell line JY, activated with PMA and ionomycin for 1, 6 hpooled (B103); NK 20 clones pooled, resting (K100); NK 20 clones pooled,activated with PMA and ionomycin for 6 h (K101); NKL clone, derived fromperipheral blood of LGL leukemia patient, IL-2 treated (K106);hematopoietic precursor line TF1, activated with PMA and ionomycin for1, 6 h pooled (C100); U937 premonocytic line, resting (M100); U937premonocytic line, activated with PMA and ionomycin for 1, 6 h pooled(M101);

[0176] elutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for 1,2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated with LPS,IFNγ, IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriated monocytes,activated with LPS, IFNγ, anti-IL-10 for 4, 16 h pooled (M106);elutriated monocytes, activated with LPS, IFNγ, IL-10 for 4, 16 h pooled(M107); elutriated monocytes, activated LPS for 1 h (M108); elutriatedmonocytes, activated LPS for 6 h (M109); DC 70% CD1a+, from CD34+GM-CSF, TNF_(—) 12 days, resting (D101); DC 70% CD1a+, from CD34+GM-CSF, TNF_(—) 12 days, activated with PMA and ionomycin for 1 hr(D102); DC 70% CD1a+, from CD34+ GM-CSF, TNF_(—) 12 days, activated withPMA and ionomycin for 6 hr (D103); DC 95% CD1a+, from CD34+ GM-CSF,TNF_(—) 12 days FACS sorted, activated with PMA and ionomycin for 1, 6 hpooled (D104); DC 95% CD14+, ex CD34+ GM-CSF, TNF_(—) 12 days FACSsorted, activated with PMA and ionomycin 1, 6 hr pooled (D105); DC CD1a+CD86+, from CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMAand ionomycin for 1, 6 h pooled (D106); DC from monocytes GM-CSF, IL-4 5days, resting (D107); DC from monocytes GM-CSF, IL-4 5 days, resting(D108); DC from monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 hpooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activated TNFα,monocyte supe for 4, 16 h pooled (D110); epithelial cells, unstimulated;epithelial cells, IL-1β activated; lung fibroblast sarcoma line MRC5,activated with PMA and ionomycin for 1, 6 h pooled (C101); kidneyepithelial carcinoma cell line CHA, activated with PMA and ionomycin for1, 6 h pooled (C102).

[0177] A rodent counterpart, e.g., mouse, has been identified, and itsdistributions will be similarly evaluated. Samples for mouse mRNAisolation can include: resting mouse fibroblastic L cell line (C200);Braf:ER (Braf fusion to estrogen receptor) transfected cells, control(C201); Mel14+ naive T cells from spleen, resting (T209); Mel14+ naive Tcells from spleen, stimulated with IFN_, IL-12, and anti IL-4 topolarize to TH1 cells, exposed to IFNγ and IL-4 for 6, 12, 24 h, pooled(T210); Mel14+ naive T cells from spleen, stimulated with IL-4 and antiIFNγ to polarize to Th2 cells, exposed to IL-4 and anti IFNγ for 6, 13,24 h, pooled (T211); T cells, TH1 polarized (Mel14 bright, CD4+ cellsfrom spleen, polarized for 7 days with IFN-_ and anti IL-4; T200); Tcells, TH2 polarized (Mel14 bright, CD4+ cells from spleen, polarizedfor 7 days with IL-4 and anti-IFN-_; T201); T cells, highly TH1polarized 3× from transgenic Balb/C (see Openshaw, et al. (1995) J. Exp.Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 24 h pooled;T202); T cells, highly TH2 polarized 3× from transgenic Balb/C(activated with anti-CD3 for 2, 6, 24 h pooled (T203); T cells, highlyTH1 polarized 3× from transgenic C57 b1/6 (activated with anti-CD3 for2, 6, 24 h pooled; T212); T cells, highly TH2 polarized 3× fromtransgenic C57 b1/6 (activated with anti-CD3 for 2, 6, 24 h pooled;T213); T cells, highly TH1 polarized (naive CD4+ T cells from transgenicBalb/C, polarized 3× with IFN_, IL-12, and anti-IL-4; stimulated withIGIF, IL-12, and anti IL-4 for 6, 12, 24 h, pooled); CD44− CD25+ pre Tcells, sorted from thymus (T204); TH1 T cell clone D1.1, resting for 3weeks after last stimulation with antigen (T205); TH1 T cell clone D1.1,10 _g/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, restingfor 3 weeks after last stimulation with antigen (T207); TH2 T cell cloneCDC35, 10 _g/ml ConA stimulated 15 h (T208); unstimulated B cell lineCH12 (B201); unstimulated mature B cell leukemia cell line A20 (B200);unstimulated large B cells from spleen (B202); B cells from totalspleen, LPS activated (B203); metrizamide enriched dendritic cells fromspleen, resting (D200); dendritic cells from bone marrow, resting(D201); unstimulated bone marrow derived dendritic cells depleted withanti B220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4(D202); bone marrow derived dendritic cells depleted with anti B220,anti CD3, and anti Class II, cultured in GM-CSF and IL-4, stimulatedwith anti CD40 for 1, 5 d, pooled (D203); monocyte cell line RAW 264.7activated with LPS 4 h (M200); bone-marrow macrophages derived with GMand M-CSF (M201); bone-marrow macrophages derived with GM-CSF,stimulated with LPS, IFN_, and IL-10 for 24 h (M205); bone-marrowmacrophages derived with GM-CSF, stimulated with LPS, IFN_, and antiIL-10 for 24 h (M206); peritoneal macrophages (M207); macrophage cellline J774, resting (M202); macrophage cell line J774+LPS +anti-IL-10 at0.5, 1, 3, 6, 12 h pooled (M203); macrophage cell line J774+LPS+IL-10 at0.5, 1, 3, 5, 12 h pooled (M204); unstimulated mast cell lines MC-9 andMCP-12 (M208); immortalized endothelial cell line derived from brainmicrovascular endothelial cells, unstimulated (E200); immortalizedendothelial cell line derived from brain microvascular endothelialcells, stimulated overnight with TNFα (E201); immortalized endothelialcell line derived from brain microvascular endothelial cells, stimulatedovernight with TNFα (E202); immortalized endothelial cell line derivedfrom brain microvascular endothelial cells, stimulated overnight withTNFA and IL-10 (E203); total aorta from wt C57 b1/6 mouse; total aortafrom 5 month ApoE KO mouse (X207); total aorta from 12 month ApoE KOmouse (X207); wt thymus (0214); total thymus, rag-1 (0208); totalkidney, rag-1 (0209); total kidney, NZ B/W mouse; and total heart, rag-1(0202). High signal was detected in the monocyte cell line RAW 264.7activated with LPS 4 h (M200); T cells, highly TH1 polarized 3× fromtransgenic C57 b1/6 (activated with anti-CD3 for 2, 6, 24 h pooled;T212); and T cells, highly TH1 polarized (naive CD4+ T cells fromtransgenic Balb/C, polarized 3× with IFNγ, IL-12, and anti-IL-4;stimulated with IGIF, IL-12, and anti IL-4 for 6, 12, 24 h, pooled).

[0178] IV. Chromosome Mapping of IL-D80

[0179] An isolated cDNA encoding the IL-D80 is used. Chromosome mappingis a standard technique. See, e.g., BIOS Laboratories (New Haven, Conn.)and methods for using a mouse somatic cell hybrid panel with PCR. Thehuman IL-D80 gene is located on chromosome 16p11.

[0180] V. Expression and Purification of IL-D80 or IL-27 Proteins

[0181] Multiple transfected cell lines are screened for one whichexpresses the cytokine at a high level compared with other cells.Various cell lines are screened and selected for their favorableproperties in handling. Natural IL-D80 can be isolated from naturalsources, or by expression from a transformed cell using an appropriateexpression vector. Purification of the expressed protein is achieved bystandard procedures, or may be combined with engineered means foreffective purification at high efficiency from cell lysates orsupernatants. FLAG or His₆ segments can be used for such purificationfeatures. Alternatively, affinity chromatography may be used withspecific antibodies, see below.

[0182] cDNAs encoding full length human and mouse IL-D80 were clonedinto the pCDM8-etag vector via HindIII-XhoI (h/mp28-E). EBI3: human andmouse EBI3 were cloned into pME18S-Ig vector via EcoRI/XhoI (h/mEBI3-Ig)and the mature portion of human EBI3 into pFlagCMV-1 vector viaHindIII-NotI (F-hEBI3). One chain fusions EBI3/p28: HindIII-XbaIfragments were generated encoding the mature part of human or mouseEBI3, followed by the synthetic linker GSGSGGSGGSGSGKL and by the maturecoding sequence of human or mouse IL-D80 via HindIII-NotI. Fragmentswere inserted into pFLAG-CMV-1 (Sigma) using HindIII-NotI sites.

[0183] WSX-1/TCCR: the preprotrypsin leader peptide and the flagtagencoding part of pFlagCMV-1 vector were deleted by PCR, instead anRGSH₆-tag was introduced via SalI/SmaI (pCMV-1-RGSH₆); the cDNA encodingthe extracellular part of human WSX-1 was cloned into this vector viaHindIII-SalI (soluble hWSX-1-R). In general restriction sites wereintroduced through the respectively used PCR primers and cDNA wasampified using standard PCR protocols. Proteins were produced viatransient expression in HEK293T cells. For experiments requiring pureproteins purification was performed by affinity chromatography using therespective protein tags.

[0184] VI. Transient Transfection, Metabolic Labeling andImmunoprecipitation.

[0185] 1×10⁶ HEK293T cells were transiently transfected with a totalamount of 5 μg plasmid DNA (control vector, expression vectors encodingh/m p28-E, F-hEBI3 and mEBI3-Ig, or respective combinations). Cells werecultured for 24 hr after transfection, then metabolically labeled for 16hr with 50 μCi/ml Pro-mix L-[³⁵S] in vitro cell labeling mix (AmershamPharmacia) in cysteine/methionine free MEM. Proteins were precipitatedfrom supernatants with either anti-Flag M2 agarose (Sigma), withanti-etag mAb bound to protein G sepharose (Amersham Pharmacia), or withprotein A sepharose (Amersham Pharmacia).

[0186] VII. Retroviral Constructs

[0187] The mature part of human and mouse WSX-1 was cloned into pMXvector via HindIII-NotI, then a sequence encoding the preprotrypsinleader peptide fused to a flag epitope was cloned into the vector inframe and 5′ of WSX-1 via BamHI-HindIII (F-h/mWSX-1). Retrovirusobtained by transfection of BOSC23 cells was used to infect parentalBa/F3 cells and cell surface expression of the desired proteins wasmonitored using a flag-PE-staining in FACS analysis.

[0188] VIII. Isolation of Homologous IL-D80 Genes

[0189] The IL-D80 cDNA, or other species counterpart sequence, can beused as a hybridization probe to screen a library from a desired source,e.g., a primate cell cDNA library. Many different species can bescreened both for stringency necessary for easy hybridization, and forpresence using a probe. Appropriate hybridization conditions will beused to select for clones exhibiting specificity of cross hybridization.

[0190] Screening by hybridization using degenerate probes based upon thepeptide sequences will also allow isolation of appropriate clones.Alternatively, use of appropriate primers for PCR screening will yieldenrichment of appropriate nucleic acid clones.

[0191] Similar methods are applicable to isolate either species,polymorphic, or allelic variants. Species variants are isolated usingcross-species hybridization techniques based upon isolation of a fulllength isolate or fragment from one species as a probe.

[0192] Alternatively, antibodies raised against human IL-D80 or IL-27will be used to screen for cells which express cross-reactive proteinsfrom an appropriate, e.g., cDNA library. The purified protein or definedpeptides are useful for generating antibodies by standard methods, asdescribed above. Synthetic peptides or purified protein are presented toan immune system to generate monoclonal or polyclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. The resulting antibodies are used for screening,purification, or diagnosis, as described.

[0193] IX. Preparation of Antibodies Specific for IL-D80 or IL-27

[0194] Synthetic peptides or purified protein are presented to an immunesystem to generate monoclonal or polyclonal antibodies. See, e.g.,Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlowand Lane (1989) Antibodies: A Laboratory Manual Cold Spring HarborPress. Polyclonal serum, or hybridomas may be prepared. In appropriatesituations, the binding reagent is either labeled as described above,e.g., fluorescence or otherwise, or immobilized to a substrate forpanning methods. Immunoselection, absorptions, and related techniquesare available to prepare selective reagents, e.g., exhibiting thedesired spectrum of selectivity for binding.

[0195] X. Generation and Analysis of Genetically Altered Animals

[0196] Transgenic mice can be generated by standard methods. Suchanimals are useful to determine the effects of deletion of the gene, inspecific tissues, or completely throughout the organism. Such mayprovide interesting insight into development of the animal or particulartissues in various stages. Moreover, the effect on various responses tobiological stress can be evaluated. See, e.g., Hogan, et al. (1995)Manipulating the Mouse Embryo: A Laboratory Manual (2d ed.) Cold SpringHarbor Laboratory Press.

[0197] IX. Expression/Distribution of IL-27

[0198] cDNAs from various libraries or cultured macrophages anddendritic cells were prepared as described (see, e.g., Bolin, et al.(1997) J. Neurosci. 17:5493-5502) and used as templates for quantitativePCR. 50 ng cDNA was analyzed for expression of human and mouse p28 andEBI3 by the fluorogenic 5′-nuclease PCR assay (see, e.g., Holland, etal. (1991) Proc. Natl. Acad. Sci. 88:7276-7280) using the ABI Prism 7700Sequence Detection System (Perkin-Elmer, Foster City, Calif.). Analysisof cDNA samples was corrected for expression of 18S rRNA using a VIClabeled probe (Perkin-Elmer) in multiplex reactions.

[0199] Analysis of a large panel of human and mouse cDNA libraries byreal time quantitative PCR showed that expression of IL-D80 and EBI3 ishighly restricted. Both mRNAs are primarily found in cells of myeloidlineage in human as well as mouse. Highest levels of human mRNA's werefound in LPS activated monocytes and monocyte derived dendritic cells(DCs). A very high level of hEBI3 mRNA but not hp28, was seen inplacenta. This observation is in agreement with earlier reports of highlevels of EBI3 protein in placental syncytiotrophoblasts [Devergne, 1997#3]. A similar pattern emerged when we analyzed the expression profileof mouse IL-D80 and EBI3. Although mEBI3 was also expressed in some Tand B cell libraries, highest levels of both mIL-D80 and mEBI3 was inactivated macrophages.

[0200] Since antigen presenting cells are also the primary source ofIL-12 (see, e.g., Macatonia, et al. (1995) J. Immunol. 154:5071-5079) westudied the kinetics of production of IL-12p35, IL-12p40, IL-D80 andEBI3 by monocyte derived DCs stimulated with LPS. Human monocytes wereisolated from peripheral blood, stimulated with GM-CSF and IL-4 for 7days to obtain immature DCs. Subsequently, these CD14+CD11c+ DCs wereactivated by LPS for various time intervals and mRNA levels of IL-12p35,IL-12p40, IL-D80 and EBI3 were analyzed by real time quantitative PCR.Despite substantial variations in the absolute amounts of PCR productfrom donor to donor and from protein to protein, the kinetics recordedwere consistent and revealed subtle differences between the fourinvestigated proteins. After an initial lag phase, message levels forIL-12p35 and IL-12p40 rapidly increased and consistently peaked between8 and 14 hours of LPS stimulation, then dropped back to background levelafter 24 hours. The profiles for the two subunits of IL-12 areessentially superimposable. A very transient expression was alsoobserved for IL-D80, although maximal message levels were already foundafter 3-6 hours. Similar to IL-12, mRNA levels for p28 declined tobackground levels after 24 hours. In contrast, EB13 showed lesstransient expression although its transcription was also rapidly inducedas early as 3h after LPS stimulus. Reaching maximal EBI3 mRNA levelsbetween 12 and 24 hours, after 72 hours EBI3 message in all three donorswas still above the unstimulated background levels.

[0201] X. Transient Transfection, Metabolic Labeling, andImmunoprecipitation

[0202] Appropriate host cells were transiently transfected with emptyvectors or expression vectors encoding hIL-D80E (E=E-tagged) and/orFhEBI3 (F=Flag-tagged). Cells were cultured to 24 hrs. and thenmetabolically labeled for 16 hrs with 50 μCi/ml PRO-MIX L-[³⁵S] in vitrocell labeling mix (Amersham Pharmacia) in cysteine/methionine free MEMcell culture media. Proteins were precipitated from 300 mL supernatantwith either the anti-His5 mAb or anti-E or anti-F mAb. The IL-12R likesubunit, WSX-1/TCCR, was also detectably labeled with RGSH₆-tag(shNR30R) and immunoprecipated as above.

[0203] XI. 2D-PAGE

[0204] Purified labeled IL-27 composite cytokine or IL-27-WSX-1/TCCRcomplex were run on a nonreducing 10% NUPAGE gel in MES running buffer(Novex). Appropriate lanes were excised, reduced in sample buffercontaining DTT, laid horizontally on two-well 10% gels, and run reducedin a second dimension. One gel was silver stained (Daiichi) while theother was blotted to a PVDF membrane and developed using appropriatemAbs. It was found that hIL-80E could be co-immunoprecipitated withshNR30R in the presence of FhEBI3 using the anti-His₆ mAb.Alternatively, shNR30R could be immunoprecipated in the presence ofhIL-80E and FhEBI3 using the anti-E mAb or anti-F mAb.

[0205] XII. Biological Effects of IL-27

[0206] A. Naive Human and Mouse T Cells

[0207] CD4+CD45RB^(high) or CD4+CD45RB^(low) T cell subsets werepurified from the spleen and mesenteric lymph nodes of >6 month oldIL-10-/-C57/B6 N12 mice as described (Davidson et al., 1998). Cells werefractionated into CD4+CD45RB^(high) and CD4+CD45RB^(low) W cellpopulations by two color sorting on a FACSTAR plus (Becton Dickinson).All populations were >99% pure upon reanalysis. CD4+CD45RB^(high) orCD4+CD45RB^(low) were put into a proliferation assay with plate boundanti-CD3 (145.2C11) stimulation as described (Davidson et al., 1998).Additions to the growth media included anti-IL-2 Mab (JES6-1A12) 100μg/ml, and cytokines as indicated. Cells were incubated for 5 days in ahumidified chamber (37° C., 5% CO₂) with [³H]TdR (Amersham) added at afinal concentration of 1 μCi/well for the last 24 h of incubation.

[0208] Sorted mouse naive T cells (CD4+CD45RB and memory/activated Tcells (CD4+CD45RB^(low)) were stimulated with CD3 mAb for four days inthe presence of anti-IL-12 antibody and various amounts of mIL-27. Uponstimulation, naive T cells, but not memory T cells, showed a strongproliferative response. Proliferation was augmented by addition of IL-12at saturating levels, revealing synergy between IL-27 and IL-12 onunstimulated T cells. IL-27 was able to act as a strong expansion factorfor anti-CD3, anti-CD28 activated naive T cells in the absence of IL-12.

[0209] FACS purified CD45RA and CD45RO T cells (purity >99%) werecultured at a density of 4×10⁴ cells/well in a 96-well plate previouslycoated with anti-CD3 antibody at 10 μg/ml and soluble anti-CD28 at 1μg/ml with or without IL-26/EBI3. Anti-hIL-2 Mab 17H12 and anti-hIL-2RMab B-B10 (Diaclone) were added at 10 μg/ml where indicated. IL-27 wasalso able to induce proliferation of FACS sorted human CD45RA naive Tcells isolated from peripheral blood mononuclear cells (PBMC). Similarto the results with mouse naive T cells, IL-27 induced strongproliferation of CD3/CD28 naive T cells in the presence of anti-IL-2.This response was enhanced by the addition of IL-12. No response wasseen with IL-27 treated CD45RO memory cells.

[0210] Thus, IL-27 dependent proliferation can be enhanced bycostimulatory signals through either CD28 or the IL-12 receptors. IL-27induced proliferation is dependent on simultaneous crosslinking ofCD3/TCR, since no proliferation was observed in the absence of CD3activation (data not shown). The same maximal proliferative responsecould be induced by stimulation with conditioned medium of p28/EB13co-transfected cells (data not shown). To compare the abilities of IL-27and IL-12 to induce proliferation of naive CD4+ T cells, FACS sortedmouse CD4+ CD45Rbhigh T cells were pre-cultured with plate boundanti-CD3 mAb, and either IL-27 or IL-12 were titrated into the cultures.IL-27 proved to be a much more potent proliferative stimulus for thesecells (FIG. 4C).

[0211] Thus, IL-27 dependent proliferation can be enhanced bycostimulatory signals through either CD28 or the IL-12 receptors. IL-27induced proliferation is dependent on simultaneous crosslinking ofCD3/TCR, since no proliferation was observed in the absence of CD3activation. The same maximal proliferative response could be induced bystimulation with conditioned medium of IL-27 co-transfected cells. Tocompare the abilities of IL-27 and IL-12 to induce proliferation ofnaive CD4+T cells, FACS sorted mouse CD4+CD45RB T cells werepre-cultured with plate bound anti-CD3 mab, and either IL-27 or IL-12were titrated into the cultures. IL-27 proved to be a much more potentproliferative stimulus for these cells.

[0212] B. Induction of IFN-γ

[0213] The ability of human and mouse IL-27 to induce the production ofIFNγ_in the presence of a neutralizing anti-IL-2 mAb, with costimulationvia anti-CD3 or anti-CD3/anti-CD28 and both in the absence and presenceof IL-12 was measured. In this assay neither hIL-27 nor hIL-12 by itselfinduced IFNγ_production in anti-CD3 or anti-CD3/anti-CD28 activatedCD4+CD45RA T cells. IFNγ production was only observed in the presence ofboth cytokines indicating strong synergy between IL-27 and IL-12.

[0214] Sorted mouse CD4+CD45RB^(high) naive T cells were stimulated for4 days with anti-CD3 mAb alone or with anti-CD3 mAb/anti-CD28 mAb andsaturating amounts of IL-27 and IL-12. In the absence of anti-CD28costimulation neither IL-27 nor IL-12 by itself was capable of inducingsubstantial amounts of IFNγ_. However, the combination of IL-27 andIL-12 induced up to about 300 ng/ml of IFNγ_. With anti-CD3/anti-CD28costimulation, IL-27 as well as IL-12 were capable of inducing IFNγproduction. The combination of both factors led to an additive effectwith IFNγ levels up to 550 ng/ml.

[0215] C. IL-27 Does Not Drive Th2 Polarization of Naive T Cells

[0216] Sorted mouse CD4+CD45RB^(high) T cells were cultured with platebound anti-CD3 and anti-CD28 in the presence of IL-4 and IL-27.Including IL-27 in the cultures led to a decreased IL-13 production bothin the absence and presence of IL-4. Thus, while inducing a strong Th1response, IL-27 does not appear to promote Th2 polarization.

[0217] D. IL-27 Binds to WSX-1/TCCR

[0218] Because of the relationship between IL-27 and the IL-6/IL-12family, the search for the signaling receptors was concentrated on thisfamily. Members of this family were introduced into BaF3 cells andtested for binding to IL-27. Of the receptors tested only Ba/F3 cellsexpressing the orphan cytokine receptor WSX-1/TCCR (see, e.g., Sprecher,et al. (1998) Biochem. Biophys, Res. Comm. 246:82-90; and Chen, et al.(2000) Nature 407:916-920) showed binding to tagged IL-27. BaF3 cellsinfected with retroviral constructs expressing either F-tagged human ormouse WSX-1 cDNA (E-hWSX-1 or F-mWSX-1) showed cellular staining usinganti-Flag mAb. Cells expressing F-hWSX-1 were then incubated with eitherhEBI3-Ig alone or with coexpressed hIL-D80-E and EBI3-Ig for tow hours.Heterodimeric IL-D80/EBI3 bound to WSX-1 while EB133-Ig itself showed nodetectable binding. Similarly, only the combination of mIL-D80-E andmEBI3-Ig provided a detectable interaction with mWSX-1-expressing BaF3cells, whereas the two individual proteins were not able to do so.Incubation of independently expressed mIL-D80-E and mEBI3-Ig withF-mWSX-1 expressing BaF3 cells also led to cellular staining.Untransfected control cells were not stained by IL-D80/EBI3,demonstrating the specificity of the observed interactions.

[0219] These results were confirmed by co-immunoprecipitationexperiments using a soluble extracellular form of hWSX-1 with aC-terminal RSGH₆-tag (R). Proteins from supernatants of transientlytransfected HEK293T cells containing F-hEBI3 or coexpressedhILD80-E/F-hEBI3 were immunoprecipitated using either Flag M2-agarose,protein G sepharose-coupled anti-etag mAb or protein G sepharose-coupledanti-H₅ mAb. The primary pricipitates were washed and then incubatedwith HEK293T cell supernatants containing shWSX-1-R. Secondaryprecipitates were separated by SDS-PAGE and subjected to western blot.Precipitaited proteins were visualized by ECL using antibodies againstthe respective protein tags. Only when all three proteins were present(hIL-D80-E, F-hEBI3 and shWSX-1-R), immunoprecipitation of one proteinbrought down both other components independently of theimmunoprecipitating antibody used. The same co-immunoprecipitationexperiment using the respective mouse orthologues had similar results.

[0220] To address the question if WSX-1 was sufficient to mediate IL-27signal transduction, proliferation of BaF3 cells expressing human ormouse WSX-1 was tested. These cells proliferate in response to IL-3 butdid not proliferate in response to IL-27. Thus WSX-1 appears to berequired but not sufficient for IL-27 mediated signal transduction. Theidentification of additional IL-27 signal transducing receptor subunitsis currently in progress.

[0221] All references cited herein are incorporated herein by referenceto the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety for all purposes.

[0222] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1 13 1 1213 DNA Homo sapiens 1 cactggccca cgctgaagat aggggacttgagttccagtc ttccttctgc taccgaccgg 60 ctttgtgacc ttgaacaaga cttcccctccctgattccat cctcatgtca catctgaagc 120 ctccaacttc tgtcactgag ctcaggattcccaggcaagc ccacggagtg ccccacaggg 180 tcagagccgt aacaggactt ggaaaataacccgaaaattg ggctcagcct gttgctgctt 240 cccttgctcc tggttcaagc tggtgtctggggattcccaa ggcccccagg gaggccccag 300 ctgagcctgc aggagctgcg gagggagttcacagtcagcc tgcatctcgc caggaagctg 360 ctctccgagg ttcggggcca ggcccaccgctttgcggaat ctcacctgcc aggagtgaac 420 ctgtacctcc tgcccctggg agagcagctccctgatgttt ccctgacctt ccaggcctgg 480 cgccgcctct ctgacccgga gcgtctctgcttcatctcca ccacgcttca gcccttccat 540 gccccgctgg gagggctggg gacccagggccgctggacca acatggagag gatgcagctg 600 tgggccatga ggctggacct ccgcgatctgcagcggcacc tccgcttcca ggtgctggct 660 gcaggattca acctcccgga ggaggaggaggaggaagagg aggaggagga ggaggagagg 720 aaggggctgc tcccaggggc actgggcagcgccttacagg gcccggccca ggtgtcctgg 780 ccccagctcc tctccaccta ccgcctgctgcactccttgg agctcgtctt atctcgggcc 840 gtgcgggagt tgctgctgct gtccaaggctgggcactcag tctggccctt ggggttccca 900 acattgagcc cccagccctg atcggtggcttcttagcccc ctgcccccca ccctttagaa 960 ctttaggact ggagtcttgg catcagggcagccttcgcat catcagcctt ggacaaggga 1020 gggctcttcc agccccctgc cccaggccctacccagtaac tgaaagcccc tctggtcctc 1080 gccagctatt tatttcttgg atatttatttattgtttagg gagatgatgg tttatttatt 1140 gtcttggggc ccgatggtcc tcctcgggccaagcccccat gctgggtgcc caataaagca 1200 ctctcatcca aaa 1213 2 242 PRT Homosapiens 2 Gln Asp Leu Glu Asn Asn Pro Lys Ile Gly Leu Ser Leu Leu LeuLeu 1 5 10 15 Pro Leu Leu Leu Val Gln Ala Gly Val Trp Gly Phe Pro ArgPro Pro 20 25 30 Gly Arg Pro Gln Leu Ser Leu Gln Glu Leu Arg Arg Glu PheThr Val 35 40 45 Ser Leu His Leu Ala Arg Lys Leu Leu Ser Glu Val Arg GlyGln Ala 50 55 60 His Arg Phe Ala Glu Ser His Leu Pro Gly Val Asn Leu TyrLeu Leu 65 70 75 80 Pro Leu Gly Glu Gln Leu Pro Asp Val Ser Leu Thr PheGln Ala Trp 85 90 95 Arg Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe Ile SerThr Thr Leu 100 105 110 Gln Pro Phe His Ala Pro Leu Gly Gly Leu Gly ThrGln Gly Arg Trp 115 120 125 Thr Asn Met Glu Arg Met Gln Leu Trp Ala MetArg Leu Asp Leu Arg 130 135 140 Asp Leu Gln Arg His Leu Arg Phe Gln ValLeu Ala Ala Gly Phe Asn 145 150 155 160 Leu Pro Glu Glu Glu Glu Glu GluGlu Glu Glu Glu Glu Glu Glu Arg 165 170 175 Lys Gly Leu Leu Pro Gly AlaLeu Gly Ser Ala Leu Gln Gly Pro Ala 180 185 190 Gln Val Ser Trp Pro GlnLeu Leu Ser Thr Tyr Arg Leu Leu His Ser 195 200 205 Leu Glu Leu Val LeuSer Arg Ala Val Arg Glu Leu Leu Leu Leu Ser 210 215 220 Lys Ala Gly HisSer Val Trp Pro Leu Gly Phe Pro Thr Leu Ser Pro 225 230 235 240 Gln Pro3 1098 DNA Mus musculus misc_feature (1)..(1) Unidentified base. 3nccaagntgg tacgcctgca ggtaccggtc cggaattccc gggtcgaccc acgcgtccgg 60ggccaggtga caggagacct tggctggcga ggactggaca ggcaacctgg ccaggagcag 120gactaaacag acaaatgaag agtgtagagg gaagaggctg agaaccgagg acagtcagag 180gaacggcaca ggggagctgg gctcagcctg ttgctgctac ccttgcttct ggtacaagct 240ggttcctggg ggttcccaac agaccccctg agccttcaag agctgcgcag ggaattcaca 300gtcagcctgt accttgccag gaagctgctc tctgaggttc agggctatgt ccacagcttt 360gctgaatctc gattgccagg agtgaacctg gacctcctgc ccctgggata ccatcttcct 420aatgtttccc tgactttcca ggcatggcat cacctctctg actctgagag actctgcttc 480ctcgctacca cacttcggcc cttccttgcc atgctgggag ggctggggac ccaggggacc 540tggaccaaca tcaagaggat gcagcaatgg agactctctc tggttcttga tgtggccctg 600tgtgtctttc gctcacaggt gctggctgca ggattcaaat gttcaaagga ggaggaggac 660aaggaggaag aggaagagga ggaagaagaa gaaaagaagc tgcccctagg gcgtctgggt 720ggccccaatc aggtgtcatc ccaagtgtcc tggccccagc tgctctatac ctaccagctc 780cttcactcca tggagcttgt cctgtctcgg gctgttcggg acctgctgct gctgtccctg 840cccaggcgcc caggctcagc cttggagttc ctaacaccta gcttcaagcc ctgatggagt 900gaccttccag ctccctccct cgcccgttaa gactctaagg ctggagtctg gccaatcaca 960ggacaggctc tagctcgttt gccttagacc aggcagggtt tcactagctc ccagccctga 1020cccaataatt taaaagccct ccagtcctta ccagatattt atttcttgga tatttattta 1080tttttaagaa atggttta 1098 4 231 PRT Mus musculus 4 Gly Leu Ser Leu LeuLeu Leu Pro Leu Leu Leu Val Gln Ala Gly Ser 1 5 10 15 Trp Gly Phe ProThr Asp Pro Leu Ser Leu Gln Glu Leu Arg Arg Glu 20 25 30 Phe Thr Val SerLeu Tyr Leu Ala Arg Lys Leu Leu Ser Glu Val Gln 35 40 45 Gly Tyr Val HisSer Phe Ala Glu Ser Arg Leu Pro Gly Val Asn Leu 50 55 60 Asp Leu Leu ProLeu Gly Tyr His Leu Pro Asn Val Ser Leu Thr Phe 65 70 75 80 Gln Ala TrpHis His Leu Ser Asp Ser Glu Arg Leu Cys Phe Leu Ala 85 90 95 Thr Thr LeuArg Pro Phe Leu Ala Met Leu Gly Gly Leu Gly Thr Gln 100 105 110 Gly ThrTrp Thr Asn Ile Lys Arg Met Gln Gln Trp Arg Leu Ser Leu 115 120 125 ValLeu Asp Val Ala Leu Cys Val Phe Arg Ser Gln Val Leu Ala Ala 130 135 140Gly Phe Lys Cys Ser Lys Glu Glu Glu Asp Lys Glu Glu Glu Glu Glu 145 150155 160 Glu Glu Glu Glu Glu Lys Lys Leu Pro Leu Gly Arg Leu Gly Gly Pro165 170 175 Asn Gln Val Ser Ser Gln Val Ser Trp Pro Gln Leu Leu Tyr ThrTyr 180 185 190 Gln Leu Leu His Ser Met Glu Leu Val Leu Ser Arg Ala ValArg Asp 195 200 205 Leu Leu Leu Leu Ser Leu Pro Arg Arg Pro Gly Ser AlaLeu Glu Phe 210 215 220 Leu Thr Pro Ser Phe Lys Pro 225 230 5 732 DNAHomo sapiens 5 atgggccaga cggcaggcga ccttggctgg cggctcagcc tgttgctgcttcccttgctc 60 ctggttcaag ctggtgtctg gggattccca aggcccccag ggaggccccagctgagcctg 120 caggagctgc ggagggagtt cacagtcagc ctgcatctcg ccaggaagctgctctccgag 180 gttcggggcc aggcccaccg ctttgcggaa tctcacctgc caggagtgaacctgtacctc 240 ctgcccctgg gagagcagct ccctgatgtt tccctgacct tccaggcctggcgccgcctc 300 tctgacccgg agcgtctctg cttcatctcc accacgcttc agcccttccatgccccgctg 360 ggagggctgg ggacccaggg ccgctggacc aacatggaga ggatgcagctgtgggccatg 420 aggctggacc tccgcgatct gcagcggcac ctccgcttcc aggtgctggctgcaggattc 480 aacctcccgg aggaggagga ggaggaagag gaggaggagg aggaggagaggaaggggctg 540 ctcccagggg cactgggcag cgccttacag ggcccggccc aggtgtcctggccccagctc 600 ctctccacct accgcctgct gcactccttg gagctcgtct tatctcgggccgtgcgggag 660 ttgctgctgc tgtccaaggc tgggcactca gtctggccct tggggttcccaacattgagc 720 ccccagccct ga 732 6 243 PRT Homo sapiens 6 Met Gly GlnThr Ala Gly Asp Leu Gly Trp Arg Leu Ser Leu Leu Leu 1 5 10 15 Leu ProLeu Leu Leu Val Gln Ala Gly Val Trp Gly Phe Pro Arg Pro 20 25 30 Pro GlyArg Pro Gln Leu Ser Leu Gln Glu Leu Arg Arg Glu Phe Thr 35 40 45 Val SerLeu His Leu Ala Arg Lys Leu Leu Ser Glu Val Arg Gly Gln 50 55 60 Ala HisArg Phe Ala Glu Ser His Leu Pro Gly Val Asn Leu Tyr Leu 65 70 75 80 LeuPro Leu Gly Glu Gln Leu Pro Asp Val Ser Leu Thr Phe Gln Ala 85 90 95 TrpArg Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe Ile Ser Thr Thr 100 105 110Leu Gln Pro Phe His Ala Pro Leu Gly Gly Leu Gly Thr Gln Gly Arg 115 120125 Trp Thr Asn Met Glu Arg Met Gln Leu Trp Ala Met Arg Leu Asp Leu 130135 140 Arg Asp Leu Gln Arg His Leu Arg Phe Gln Val Leu Ala Ala Gly Phe145 150 155 160 Asn Leu Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu GluGlu Glu 165 170 175 Arg Lys Gly Leu Leu Pro Gly Ala Leu Gly Ser Ala LeuGln Gly Pro 180 185 190 Ala Gln Val Ser Trp Pro Gln Leu Leu Ser Thr TyrArg Leu Leu His 195 200 205 Ser Leu Glu Leu Val Leu Ser Arg Ala Val ArgGlu Leu Leu Leu Leu 210 215 220 Ser Lys Ala Gly His Ser Val Trp Pro LeuGly Phe Pro Thr Leu Ser 225 230 235 240 Pro Gln Pro 7 991 DNA Musmusculus misc_feature (1)..(1) 7 atgggccaga cggcaggcga ccttggctggcggctcagcc tgttgctgct acccttgctt 60 ctggtacaag ctggttcctg ggggttcccaacagaccccc tgagccttca agagctgcgc 120 agggaattca cagtcagcct gtaccttgccaggaagctgc tctctgaggt tcagggctat 180 gtccacagct ttgctgaatc tcgattgccaggagtgaacc tggacctcct gcccctggga 240 taccatcttc ccaatgtttc cctgactttccaggcatggc atcacctctc tgactctgag 300 agactctgct tcctcgctac cacacttcggcccttccctg ccatgctggg agggctgggg 360 acccagggga cctggaccag ctcagagagggagcagctgt gggccatgag gctggatctc 420 cgggacctgc acaggcacct ccgctttcaggtgctggctg caggattcaa atgttcaaag 480 gaggaggagg acaaggagga agaggaagaggaggaagaag aagaaaagaa gctgccccta 540 ggggctctgg gtggccccaa tcaggtgtcatcccaagtgt cctggcccca gctgctctat 600 acctaccagc tccttcactc cctggagcttgtcctgtctc gggctgttcg ggacctgctg 660 ctgctgtccc tgcccaggcg cccaggctcagcctgggatt cctaacacct agcttcaagc 720 cctatggagt gaccttccag ctccctccctcgcccgttaa gactctaagg ctggagtctg 780 gccaatcaca ggacaggctc tagctcgtttgccttagacc aggcagggct tcactagctc 840 ccagccctga cccaataatt taaaagccctccagtcctta ccagatattt atttcttgga 900 tatttattta tttttaagaa atggtttatttattgtttca ctcttgagtt aggccaccat 960 gctgggtgcc taataaagcc atccagcccg g991 8 234 PRT Mus musculus 8 Met Gly Gln Thr Ala Gly Asp Leu Gly Trp ArgLeu Ser Leu Leu Leu 1 5 10 15 Leu Pro Leu Leu Leu Val Gln Ala Gly SerTrp Gly Phe Pro Thr Asp 20 25 30 Pro Leu Ser Leu Gln Glu Leu Arg Arg GluPhe Thr Val Ser Leu Tyr 35 40 45 Leu Ala Arg Lys Leu Leu Ser Glu Val GlnGly Tyr Val His Ser Phe 50 55 60 Ala Glu Ser Arg Leu Pro Gly Val Asn LeuAsp Leu Leu Pro Leu Gly 65 70 75 80 Tyr His Leu Pro Asn Val Ser Leu ThrPhe Gln Ala Trp His His Leu 85 90 95 Ser Asp Ser Glu Arg Leu Cys Phe LeuAla Thr Thr Leu Arg Pro Phe 100 105 110 Pro Ala Met Leu Gly Gly Leu GlyThr Gln Gly Thr Trp Thr Ser Ser 115 120 125 Glu Arg Glu Gln Leu Trp AlaMet Arg Leu Asp Leu Arg Asp Leu His 130 135 140 Arg His Leu Arg Phe GlnVal Leu Ala Ala Gly Phe Lys Cys Ser Lys 145 150 155 160 Glu Glu Glu AspLys Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Lys 165 170 175 Lys Leu ProLeu Gly Ala Leu Gly Gly Pro Asn Gln Val Ser Ser Gln 180 185 190 Val SerTrp Pro Gln Leu Leu Tyr Thr Tyr Gln Leu Leu His Ser Leu 195 200 205 GluLeu Val Leu Ser Arg Ala Val Arg Asp Leu Leu Leu Leu Ser Leu 210 215 220Pro Arg Arg Pro Gly Ser Ala Trp Asp Ser 225 230 9 1161 DNA Homo sapiensCDS (14)..(700) 9 gaattccgca gcc atg acc ccg cag ctt ctc ctg gcc ctt gtcctc tgg 49 Met Thr Pro Gln Leu Leu Leu Ala Leu Val Leu Trp 1 5 10 gccagc tgc ccg ccc tgc agt gga agg aaa ggg ccc cca gca gct ctg 97 Ala SerCys Pro Pro Cys Ser Gly Arg Lys Gly Pro Pro Ala Ala Leu 15 20 25 aca ctgccc cgg gtg caa tgc cga gcc tct cgg tac ccg atc gcc gtg 145 Thr Leu ProArg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val 30 35 40 gat tgc tcctgg acc ctg ccg cct gct cca aac tcc acc agc ccc gtg 193 Asp Cys Ser TrpThr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val 45 50 55 60 tcc ttc attgcc acg tac agg ctc ggc atg gct gcc cgg ggc cac agc 241 Ser Phe Ile AlaThr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser 65 70 75 tgg ccc tgc ctgcag cag acg cca acg tcc acc agc tgc acc atc acg 289 Trp Pro Cys Leu GlnGln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr 80 85 90 gat gtc cag ctg ttctcc atg gct ccc tac gtg ctc aat gtc acc gcc 337 Asp Val Gln Leu Phe SerMet Ala Pro Tyr Val Leu Asn Val Thr Ala 95 100 105 gtc cac ccc tgg ggctcc agc agc agc ttc gtg cct ttc ata aca gag 385 Val His Pro Trp Gly SerSer Ser Ser Phe Val Pro Phe Ile Thr Glu 110 115 120 cac atc atc aag cccgac cct cca gaa ggc gtg cgc cta agc ccc ctc 433 His Ile Ile Lys Pro AspPro Pro Glu Gly Val Arg Leu Ser Pro Leu 125 130 135 140 gct gag cgc cacgta cag gtg cag tgg gag cct ccc ggg tcc tgg ccc 481 Ala Glu Arg His ValGln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro 145 150 155 ttc cca gag atcttc tca ctg aag tac tgg atc cgt tac aag cgt cag 529 Phe Pro Glu Ile PheSer Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln 160 165 170 gga gct gcg cgcttc cac cgg gtg ggg ccc att gaa gcc acg tcc ttc 577 Gly Ala Ala Arg PheHis Arg Val Gly Pro Ile Glu Ala Thr Ser Phe 175 180 185 atc ctc agg gctgtg cgg ccc cga gcc agg tac tac gtc caa gtg gcg 625 Ile Leu Arg Ala ValArg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala 190 195 200 gct cag gac ctcaca gac tac ggg gaa ctg agt gac tgg agt ctc ccc 673 Ala Gln Asp Leu ThrAsp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro 205 210 215 220 gcc act gccaca atg agc ctg ggc aag tagcaagggc ttcccgctgc 720 Ala Thr Ala Thr MetSer Leu Gly Lys 225 ctccagacag cacctgggtc ctcgccaccc taagccccgggacacctgtt ggagggcgga 780 tgggatctgc ctagcctggg ctggagtcct tgctttgctgctgctgagct gccgggcaac 840 ctcagatgac cgacttttcc ctttgagcct cagtttctctagctgagaaa tggagatgta 900 ctactctctc ctttaccttt acctttacca cagtgcagggctgactgaac tgtcactgtg 960 agatattttt tattgtttaa ttagaaaaga attgttgttgggctgggcgc agtggatcgc 1020 acctgtaatc ccagtcactg ggaagccgac gtgggtgggtagcttgaggc caggagctcg 1080 aaaccagtcc gggccacaca gcaagacccc atctctaaaaaattaatata aatataaaat 1140 aaaaaaaaaa aaaaggaatt c 1161 10 229 PRT Homosapiens 10 Met Thr Pro Gln Leu Leu Leu Ala Leu Val Leu Trp Ala Ser CysPro 1 5 10 15 Pro Cys Ser Gly Arg Lys Gly Pro Pro Ala Ala Leu Thr LeuPro Arg 20 25 30 Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp CysSer Trp 35 40 45 Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser PheIle Ala 50 55 60 Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp ProCys Leu 65 70 75 80 Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr AspVal Gln Leu 85 90 95 Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala ValHis Pro Trp 100 105 110 Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr GluHis Ile Ile Lys 115 120 125 Pro Asp Pro Pro Glu Gly Val Arg Leu Ser ProLeu Ala Glu Arg His 130 135 140 Val Gln Val Gln Trp Glu Pro Pro Gly SerTrp Pro Phe Pro Glu Ile 145 150 155 160 Phe Ser Leu Lys Tyr Trp Ile ArgTyr Lys Arg Gln Gly Ala Ala Arg 165 170 175 Phe His Arg Val Gly Pro IleGlu Ala Thr Ser Phe Ile Leu Arg Ala 180 185 190 Val Arg Pro Arg Ala ArgTyr Tyr Val Gln Val Ala Ala Gln Asp Leu 195 200 205 Thr Asp Tyr Gly GluLeu Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr 210 215 220 Met Ser Leu GlyLys 225 11 2628 DNA Homo sapiens CDS (112)..(2019) 11 gtgggttcggcttcccgttg cgcctcgggg gctgtaccca gagctcgaag aggagcagcg 60 cggcccgcacccggcaaggc tgggccggac tcggggctcc cgagggacgc c atg cgg 117 Met Arg 1 ggaggc agg ggc ggc cct ttc tgg ctg tgg ccg ctg ccc aag ctg gcg 165 Gly GlyArg Gly Gly Pro Phe Trp Leu Trp Pro Leu Pro Lys Leu Ala 5 10 15 ctg ctgcct ctg ttg tgg gtg ctt ttc cag cgg acg cgt ccc cag ggc 213 Leu Leu ProLeu Leu Trp Val Leu Phe Gln Arg Thr Arg Pro Gln Gly 20 25 30 agc gcc gggcca ctg cag tgc tac gga gtt gga ccc ttg ggc gac ttg 261 Ser Ala Gly ProLeu Gln Cys Tyr Gly Val Gly Pro Leu Gly Asp Leu 35 40 45 50 aac tgc tcgtgg gag cct ctt ggg gac ctg gga gcc ccc tcc gag tta 309 Asn Cys Ser TrpGlu Pro Leu Gly Asp Leu Gly Ala Pro Ser Glu Leu 55 60 65 cac ctc cag agccaa aag tac cgt tcc aac aaa acc cag act gtg gca 357 His Leu Gln Ser GlnLys Tyr Arg Ser Asn Lys Thr Gln Thr Val Ala 70 75 80 gtg gca gcc gga cggagc tgg gtg gcc att cct cgg gaa cag ctc acc 405 Val Ala Ala Gly Arg SerTrp Val Ala Ile Pro Arg Glu Gln Leu Thr 85 90 95 atg tct gac aaa ctc cttgtc tgg ggc act aag gca ggc cag cct ctc 453 Met Ser Asp Lys Leu Leu ValTrp Gly Thr Lys Ala Gly Gln Pro Leu 100 105 110 tgg ccc ccc gtc ttc gtgaac cta gaa acc caa atg aag cca aac gcc 501 Trp Pro Pro Val Phe Val AsnLeu Glu Thr Gln Met Lys Pro Asn Ala 115 120 125 130 ccc cgg ctg ggc cctgac gtg gac ttt tcc gag gat gac ccc ctg gag 549 Pro Arg Leu Gly Pro AspVal Asp Phe Ser Glu Asp Asp Pro Leu Glu 135 140 145 gcc act gtc cat tgggcc cca cct aca tgg cca tct cat aaa gtt ctg 597 Ala Thr Val His Trp AlaPro Pro Thr Trp Pro Ser His Lys Val Leu 150 155 160 atc tgc cag ttc cactac cga aga tgt cag gag gcg gcc tgg acc ctg 645 Ile Cys Gln Phe His TyrArg Arg Cys Gln Glu Ala Ala Trp Thr Leu 165 170 175 ctg gaa ccg gag ctgaag acc ata ccc ctg acc cct gtt gag atc caa 693 Leu Glu Pro Glu Leu LysThr Ile Pro Leu Thr Pro Val Glu Ile Gln 180 185 190 gat ttg gag cta gccact ggc tac aaa gtg tat ggc cgc tgc cgg atg 741 Asp Leu Glu Leu Ala ThrGly Tyr Lys Val Tyr Gly Arg Cys Arg Met 195 200 205 210 gag aaa gaa gaggat ttg tgg ggc gag tgg agc ccc att ttg tcc ttc 789 Glu Lys Glu Glu AspLeu Trp Gly Glu Trp Ser Pro Ile Leu Ser Phe 215 220 225 cag aca ccg ccttct gct cca aaa gat gtg tgg gta tca ggg aac ctc 837 Gln Thr Pro Pro SerAla Pro Lys Asp Val Trp Val Ser Gly Asn Leu 230 235 240 tgt ggg acg cctgga gga gag gaa cct ttg ctt cta tgg aag gcc cca 885 Cys Gly Thr Pro GlyGly Glu Glu Pro Leu Leu Leu Trp Lys Ala Pro 245 250 255 ggg ccc tgt gtgcag gtg agc tac aaa gtc tgg ttc tgg gtt gga ggt 933 Gly Pro Cys Val GlnVal Ser Tyr Lys Val Trp Phe Trp Val Gly Gly 260 265 270 cgt gag ctg agtcca gaa gga att acc tgc tgc tgc tcc cta att ccc 981 Arg Glu Leu Ser ProGlu Gly Ile Thr Cys Cys Cys Ser Leu Ile Pro 275 280 285 290 agt ggg gcggag tgg gcc agg gtg tcc gct gtc aac gcc aca agc tgg 1029 Ser Gly Ala GluTrp Ala Arg Val Ser Ala Val Asn Ala Thr Ser Trp 295 300 305 gag cct ctcacc aac ctc tct ttg gtc tgc ttg gat tca gcc tct gcc 1077 Glu Pro Leu ThrAsn Leu Ser Leu Val Cys Leu Asp Ser Ala Ser Ala 310 315 320 ccc cgt agcgtg gca gtc agc agc atc gct ggg agc acg gag cta ctg 1125 Pro Arg Ser ValAla Val Ser Ser Ile Ala Gly Ser Thr Glu Leu Leu 325 330 335 gtg acc tggcaa ccg ggg cct ggg gaa cca ctg gag cat gta gtg gac 1173 Val Thr Trp GlnPro Gly Pro Gly Glu Pro Leu Glu His Val Val Asp 340 345 350 tgg gct cgagat ggg gac ccc ctg gag aaa ctc aac tgg gtc cgg ctt 1221 Trp Ala Arg AspGly Asp Pro Leu Glu Lys Leu Asn Trp Val Arg Leu 355 360 365 370 ccc cctggg aac ctc agt gct ctg tta cca ggg aat ttc act gtc ggg 1269 Pro Pro GlyAsn Leu Ser Ala Leu Leu Pro Gly Asn Phe Thr Val Gly 375 380 385 gtc ccctat cga atc act gtg acc gca gtc tct gct tca ggc ttg gcc 1317 Val Pro TyrArg Ile Thr Val Thr Ala Val Ser Ala Ser Gly Leu Ala 390 395 400 tct gcatcc tcc gtc tgg ggg ttc agg gag gaa tta gca ccc cta gtg 1365 Ser Ala SerSer Val Trp Gly Phe Arg Glu Glu Leu Ala Pro Leu Val 405 410 415 ggg ccaacg ctt tgg cga ctc caa gat gcc cct cca ggg acc ccc gcc 1413 Gly Pro ThrLeu Trp Arg Leu Gln Asp Ala Pro Pro Gly Thr Pro Ala 420 425 430 ata gcgtgg gga gag gtc cca agg cac cag ctt cga ggc cac ctc acc 1461 Ile Ala TrpGly Glu Val Pro Arg His Gln Leu Arg Gly His Leu Thr 435 440 445 450 cactac acc ttg tgt gca cag agt gga acc agc ccc tcc gtc tgc atg 1509 His TyrThr Leu Cys Ala Gln Ser Gly Thr Ser Pro Ser Val Cys Met 455 460 465 aatgtg agt ggc aac aca cag agt gtc acc ctg cct gac ctt cct tgg 1557 Asn ValSer Gly Asn Thr Gln Ser Val Thr Leu Pro Asp Leu Pro Trp 470 475 480 ggtccc tgt gag ctg tgg gtg aca gca tct acc atc gct gga cag ggc 1605 Gly ProCys Glu Leu Trp Val Thr Ala Ser Thr Ile Ala Gly Gln Gly 485 490 495 cctcct ggt ccc atc ctc cgg ctt cat cta cca gat aac acc ctg agg 1653 Pro ProGly Pro Ile Leu Arg Leu His Leu Pro Asp Asn Thr Leu Arg 500 505 510 tggaaa gtt ctg ccg ggc atc cta ttc ttg tgg ggc ttg ttc ctg ttg 1701 Trp LysVal Leu Pro Gly Ile Leu Phe Leu Trp Gly Leu Phe Leu Leu 515 520 525 530ggg tgt ggc ctg agc ctg gcc acc tct gga agg tgc tac cac cta agg 1749 GlyCys Gly Leu Ser Leu Ala Thr Ser Gly Arg Cys Tyr His Leu Arg 535 540 545cac aaa gtg ctg ccc cgc tgg gtc tgg gag aaa gtt cct gat cct gcc 1797 HisLys Val Leu Pro Arg Trp Val Trp Glu Lys Val Pro Asp Pro Ala 550 555 560aac agc agt tca ggc cag ccc cac atg gag caa gta cct gag gcc cag 1845 AsnSer Ser Ser Gly Gln Pro His Met Glu Gln Val Pro Glu Ala Gln 565 570 575ccc ctt ggg gac ttg ccc atc ctg gaa gtg gag gag atg gag ccc ccg 1893 ProLeu Gly Asp Leu Pro Ile Leu Glu Val Glu Glu Met Glu Pro Pro 580 585 590ccg gtt atg gag tcc tcc cag ccc gcc cag gcc acc gcc ccg ctt gac 1941 ProVal Met Glu Ser Ser Gln Pro Ala Gln Ala Thr Ala Pro Leu Asp 595 600 605610 tct ggg tat gag aag cac ttc ctg ccc aca cct gag gag ctg ggc ctt 1989Ser Gly Tyr Glu Lys His Phe Leu Pro Thr Pro Glu Glu Leu Gly Leu 615 620625 ctg ggg ccc ccc agg cca cag gtt ctg gcc tgaaccacac gtctggctgg 2039Leu Gly Pro Pro Arg Pro Gln Val Leu Ala 630 635 gggctgccag ccaggctagagggatgctca tgcaggttgc accccagtcc tggattagcc 2099 ctcttgatgg atgaagacactgaggactca gagaggctga gtcacttacc tgaggacacc 2159 cagccaggca gagctgggattgaaggaccc ctatagagaa gggcttggcc cccatgggga 2219 agacacggat ggaaggtggagcaaaggaaa atacatgaaa ttgagagtgg cagctgcctg 2279 ccaaaatctg ttccgctgtaacagaactga atttggaccc cagcacagtg gctcacgcct 2339 gtaatcccag cactttggcaggccaaggtg gaaggatcac ttagagctag gagtttgaga 2399 ccagcctggg caatatagcaagacccctca ctanaaaaat aaaacatcaa aaacaaaaac 2459 aattagctgg gcatgatggcacacacctgt agtccgagcc acttgggagg ctgaggtggg 2519 aggatcggtt gagcccaggagttcgaagct gcagggacct ctgattgcac cactgcactc 2579 caggctgggt aacagaatgagaccttatct caaaaataaa caaactaat 2628 12 636 PRT Homo sapiensmisc_feature (2433)..(2433) Unidentified base. 12 Met Arg Gly Gly ArgGly Gly Pro Phe Trp Leu Trp Pro Leu Pro Lys 1 5 10 15 Leu Ala Leu LeuPro Leu Leu Trp Val Leu Phe Gln Arg Thr Arg Pro 20 25 30 Gln Gly Ser AlaGly Pro Leu Gln Cys Tyr Gly Val Gly Pro Leu Gly 35 40 45 Asp Leu Asn CysSer Trp Glu Pro Leu Gly Asp Leu Gly Ala Pro Ser 50 55 60 Glu Leu His LeuGln Ser Gln Lys Tyr Arg Ser Asn Lys Thr Gln Thr 65 70 75 80 Val Ala ValAla Ala Gly Arg Ser Trp Val Ala Ile Pro Arg Glu Gln 85 90 95 Leu Thr MetSer Asp Lys Leu Leu Val Trp Gly Thr Lys Ala Gly Gln 100 105 110 Pro LeuTrp Pro Pro Val Phe Val Asn Leu Glu Thr Gln Met Lys Pro 115 120 125 AsnAla Pro Arg Leu Gly Pro Asp Val Asp Phe Ser Glu Asp Asp Pro 130 135 140Leu Glu Ala Thr Val His Trp Ala Pro Pro Thr Trp Pro Ser His Lys 145 150155 160 Val Leu Ile Cys Gln Phe His Tyr Arg Arg Cys Gln Glu Ala Ala Trp165 170 175 Thr Leu Leu Glu Pro Glu Leu Lys Thr Ile Pro Leu Thr Pro ValGlu 180 185 190 Ile Gln Asp Leu Glu Leu Ala Thr Gly Tyr Lys Val Tyr GlyArg Cys 195 200 205 Arg Met Glu Lys Glu Glu Asp Leu Trp Gly Glu Trp SerPro Ile Leu 210 215 220 Ser Phe Gln Thr Pro Pro Ser Ala Pro Lys Asp ValTrp Val Ser Gly 225 230 235 240 Asn Leu Cys Gly Thr Pro Gly Gly Glu GluPro Leu Leu Leu Trp Lys 245 250 255 Ala Pro Gly Pro Cys Val Gln Val SerTyr Lys Val Trp Phe Trp Val 260 265 270 Gly Gly Arg Glu Leu Ser Pro GluGly Ile Thr Cys Cys Cys Ser Leu 275 280 285 Ile Pro Ser Gly Ala Glu TrpAla Arg Val Ser Ala Val Asn Ala Thr 290 295 300 Ser Trp Glu Pro Leu ThrAsn Leu Ser Leu Val Cys Leu Asp Ser Ala 305 310 315 320 Ser Ala Pro ArgSer Val Ala Val Ser Ser Ile Ala Gly Ser Thr Glu 325 330 335 Leu Leu ValThr Trp Gln Pro Gly Pro Gly Glu Pro Leu Glu His Val 340 345 350 Val AspTrp Ala Arg Asp Gly Asp Pro Leu Glu Lys Leu Asn Trp Val 355 360 365 ArgLeu Pro Pro Gly Asn Leu Ser Ala Leu Leu Pro Gly Asn Phe Thr 370 375 380Val Gly Val Pro Tyr Arg Ile Thr Val Thr Ala Val Ser Ala Ser Gly 385 390395 400 Leu Ala Ser Ala Ser Ser Val Trp Gly Phe Arg Glu Glu Leu Ala Pro405 410 415 Leu Val Gly Pro Thr Leu Trp Arg Leu Gln Asp Ala Pro Pro GlyThr 420 425 430 Pro Ala Ile Ala Trp Gly Glu Val Pro Arg His Gln Leu ArgGly His 435 440 445 Leu Thr His Tyr Thr Leu Cys Ala Gln Ser Gly Thr SerPro Ser Val 450 455 460 Cys Met Asn Val Ser Gly Asn Thr Gln Ser Val ThrLeu Pro Asp Leu 465 470 475 480 Pro Trp Gly Pro Cys Glu Leu Trp Val ThrAla Ser Thr Ile Ala Gly 485 490 495 Gln Gly Pro Pro Gly Pro Ile Leu ArgLeu His Leu Pro Asp Asn Thr 500 505 510 Leu Arg Trp Lys Val Leu Pro GlyIle Leu Phe Leu Trp Gly Leu Phe 515 520 525 Leu Leu Gly Cys Gly Leu SerLeu Ala Thr Ser Gly Arg Cys Tyr His 530 535 540 Leu Arg His Lys Val LeuPro Arg Trp Val Trp Glu Lys Val Pro Asp 545 550 555 560 Pro Ala Asn SerSer Ser Gly Gln Pro His Met Glu Gln Val Pro Glu 565 570 575 Ala Gln ProLeu Gly Asp Leu Pro Ile Leu Glu Val Glu Glu Met Glu 580 585 590 Pro ProPro Val Met Glu Ser Ser Gln Pro Ala Gln Ala Thr Ala Pro 595 600 605 LeuAsp Ser Gly Tyr Glu Lys His Phe Leu Pro Thr Pro Glu Glu Leu 610 615 620Gly Leu Leu Gly Pro Pro Arg Pro Gln Val Leu Ala 625 630 635 13 15 PRTSynthetic. 13 Gly Ser Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Gly LysLeu 1 5 10 15

What is claimed is:
 1. An isolated or recombinant polynucleotideencoding an antigenic polypeptide comprising at least 17 contiguousamino acids from the mature polypeptide from SEQ ID NO: 2, 4, 6, or 8.2. The polynucleotide of claim 1, encoding a mature polypeptide from SEQID NO: 2, 4, 6, or
 8. 3. The polynucleotide of claim 1, which hybridizesat 55° C., less than 500 mM salt, and 50% formamide to the codingportions of SEQ ID NO: 1, 3, 5, or
 7. 4. The polynucleotide of claim 3,comprising at least 35 contiguous nucleotides of the coding portion ofSEQ ID NO: 1, 3, 5, or
 7. 5. An expression vector comprising thepolynucleotide of claim
 1. 6. A host cell containing the expressionvector of claim 5, including a eukaryotic cell.
 7. A method of making anantigenic polypeptide comprising expressing a recombinant polynucleotideof claim
 1. 8. A method for forming a duplex with a polynucleotide ofclaim 1, comprising contacting said polynucleotide with a probe thathybridizes, under stringent conditions, to at least 25 contiguousnucleotides of the coding portion of SEQ ID NO: 1, 3, 5, or 7; therebyforming said duplex.
 9. A kit for the detection of a polynucleotide ofclaim 1, comprising a polynucleotide that hybridizes, under stringenthybridization conditions, to at least 17 contiguous nucleotides of apolynucleotide of claim
 1. 10. The kit of claim 9, wherein said probe isdetectably labeled.
 11. A binding compound comprising an antibodybinding site which specifically binds to at least 17 contiguous aminoacids from SEQ ID NO: 2, 4, 6, or
 8. 12. The binding compound of claim1, wherein: a) said antibody binding site is: 1) specificallyimmunoreactive with a polypeptide of SEQ ID NO: 2, 4, 6 or 8; 2) raisedagainst a purified or recombinantly produced human IL-D80 or IL-27protein; or 3) in a monoclonal antibody, Fab, or F(ab)2; or b) saidbinding compound is: 1) an antibody molecule; 2) a polyclonal antiserum;3) detectably labeled; 4) sterile; or 5) in a buffered composition. 13.A method using the binding compound of claim 11, comprising contactingsaid binding compound with a biological sample comprising an antigen,wherein said contacting results in formation of a bindingcompound:antigen complex.
 14. The method of claim 13, wherein saidbiological sample is from a human, and wherein said binding compound isan antibody.
 15. A detection kit comprising said binding compound ofclaim 12, and: a) instructional material for the use of said bindingcompound for said detection; or b) a compartment providing segregationof said binding compound.
 16. A substantially pure or isolated antigenicpolypeptide, which binds to said binding composition of claim 11, andfurther comprises at least 17 contiguous amino acids from SEQ ID NO: 2,4, 6, or
 8. 17. The polypeptide of claim 16, which: a) comprises atleast a fragment of at least 25 contiguous amino acid residues from aprimate IL-D80 or IL-27 protein; b) is a soluble polypeptide; c) isdelectably labeled; d) is in a sterile composition; e) is in a bufferedcomposition; f) binds to a cell surface receptor; g) is recombinantlyproduced; or h) has a naturally occurring polypeptide sequence.
 18. Thepolypeptide of claim 17, which comprises at least 17 contiguous aminoacids of SEQ ID NO: 2, 4, 6, or
 8. 19. A method of modulating physiologyor development of a cell or tissue culture cells comprising contactingsaid cell with an agonist or antagonist of a primate IL-D80 or IL-27.20. The method of claim 19, wherein: a) said contacting is incombination with an agonist or antagonist of IL-12; or b) saidcontacting is with an antagonist, including binding compositioncomprising an antibody binding site which specifically binds an IL-D80or IL-27.
 21. A composite cytokine comprising a plurality of segments ofSEQ ID NO: 2, 4, 6, or 8 and SEQ ID NO:
 10. 22. An isolated orrecombinant polynucleotide encoding the composite cytokine of claim 21.23. A binding composition which specifically binds to an antigenicfragment of the composite cytokine of claim
 21. 24. A receptorsubunit:ligand composition comprising a plurality of polypeptidesegments of SEQ ID NO: 2, 4, 6, or 8; SEQ ID NO:10; and SEQ ID NO:12.25. A binding composition which specifically binds to an antigenicfragment of the receptor